This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).

Tüm deney prosedürleri Birmingham Alabama Üniversitesi Hayvan Rehberine uygun olarak yapılmaktadır. 1. hiPSC-CM&#39;ler içine hiPSCs Farklılaşan <ol><li> Kaplayın, gece boyunca 4 ° C&#39;de önceden soğutulmuş büyüme faktörü azaltılmış jelatinimsi bir protein karışımı ile 6 delikli bir plakanın deliklerine. Kullanmadan önce, jelatinimsi protein karışımı aspire. % 5 CO2 ve 10 uM ROCK inhibitörü ile 37 ° C mTeSR1 orta ek hücreleri (oyuk başına 1 x 10 5 hücre) önceden kaplanmış plakalar üzerine hiPSCs tohum, ve kültür. </li><li> Hücreler% 90 izdiham ulaşana kadar her gün orta yenile; Daha sonra,% 5 CO2 ve 37 ° C&#39;de iki gün süre ile ortam (0.5 mg, jelatinimsi Protein 6-çukurlu plaka başına karışımı, oyuk başına 2 ml&#39;lik ortam) ve kültür hücreleri büyüme faktörü azaltılmış jelatinimsi bir protein karışımı ekleyin. <ol><li> orta değiştirmek için, yavaşça vakum yoluyla petri ile gelen orta dışarı emmekdışarı hücrelerin dokunmadan, ve bir transfer pipet kullanarak yeni orta ekleyin. </li></ol></li><li> ile desteklenmiş RPMI1640 ortam maddesi ile ortamının değiştirilmesiyle farklılık başlatın insülin olmadan jelatinimsi protein karışımı, B27 Büyüme faktörü indirgenmiş ve 100 ng / ml Aktivin A; Kültür% 5 CO2 ve 24 saat boyunca 37 ° C&#39;de hücre. </li><li> , RPMI1640 insülin olmayan B27 ile takviye edilmiş ortam, 10 ng / ml olan kemik morfojenik protein 4 (BMP-4), ve 10 ng / ml temel fibroblast büyüme faktörü (bFGF) ile orta yerine Kültür% 5 CO2 ve 96 saat boyunca 37 ° C&#39;de hücre. </li><li> B27 ile takviye edilmiş RPMI1640 ortam maddesi ile orta takın ve% 5 CO2 ve 37 ° C &#39;de kültür hücreleri devam etmektedir; her 3 günde bir orta yenileyin. </li><li> farklılaşma başlatarak 3 gün sonra ~ mikroskop altında hücrelerin yakalanma kümeleri gözlemleyin. ~ 7 gün ilk kasılmaları gözlemledikten sonra kümeleri toplayın ve Hanks Dengeli Tuz Çözeltisi yıkayın. <ol><li> hafifçe çalkalanarak 37 ° C&#39;de 10 dakika süreyle 100 U / ml kolajenaz IV ihtiva eden Hanks tamponu içerisinde onları kuluçka plaka kümelenmiş-hücreleri ayırmak; Daha sonra, 5 dakika boyunca etilendiamintetraasetik asit (EDTA),% 0.25 tripsin ekleyin. </li><li> RPMI / B27 ortamında% 10 fetal bovin serumu ile enzim çözeltisi nötralize ve RPMI / B27 ortamı içinde tekrar süspansiyon hücreleri. </li><li> hemasitometre hücrelerinin sayısını. Kültür olmayan kardiyomiyosit hücreler kültür kaplarına yüzeyine yapışır sağlayacak% 5 CO2 ve en az 3 saat 37 ° C&#39;de en az 10 cm yemekleri hücreleri. </li></ol></li><li> HiPSC-CMS içeren hücre süspansiyonu, toplamak ve jelatinli protein karışımı -kaplı yüzeye kültürleme yoluyla (10 cm çanak başına 6 ila 10 x 1 civarında hücreleri) hücreleri korumak. </li></ol> 2. hiPSC-EC içine hiPSCs Farklılaşan <ol><li> th inkübe edilerek tek hücre içine hiPSC popülasyonu ayrıştırmaları% 5 CO2 ve 5 dakika için 37 ° C &#39;de maddesi kenetleme em. Taze mTeSR1 ortamı içeren 15 ml&#39;lik bir santrifüj tüpüne hücreleri aktarın. </li><li> 5 dakika boyunca 200 xg&#39;de hücreleri aşağı doğru döndürün. 10 uM ROCK inhibitörü ile takviye edilmiş ortam içinde mTeSR1 tekrar süspansiyon hücreleri. Bir hemasitometre ile hücre yoğunluğu belirlemek. </li><li> 24 oyuklu plakanın bir oyuğuna 20 NIH birim / ml trombin çözeltisi 250 ul ekle. </li><li> 12.5 mg / ml fibrinojen çözeltisi 250 ul 1 x 10 6 hiPSCs ekleyin. Ve trombin yüklü de hücre-ihtiva eden fibrinojen çözeltisi 250 ul ilave edin. Karışım dakika içinde bir hiPSC içeren fibrin iskele oluşturacak şekilde katılaşmaya gözlemleyin. </li><li> cam kapak forseps ile bir 6-yuvalı plaka oyuklarına katılaştırılmış hücre içeren iskele aktarın ve insülin olmadan% 2 B27 ile takviye 2 mi EBM2 ortamında iskeleleri kültürlenmesiyle farklılaşma başlatılması, aktivin-A (50 ng / mi) çözündürüldü, ve BMP-4, 24 saat için (25 ng / ml),% 5 CO de <sUB&gt; 2 37 ° C. <ol><li> nazikçe hücreleri dokunmadan vakum yoluyla eski orta emerek orta değiştirin. İnsülin olmadan B27 ile takviye edilmiş yeni EBM2 orta, vasküler endotelyal büyüme faktörü (VEGF, 50 ng / ml), eritropoietin (EPO, 100 ng / ml), ve transforme edici büyüme faktörü ß1 (TGFβ1, 10 ng / ml) ve kültür ekleme % 5 iskeleleri CO2 ve 48 saat boyunca 37 ° C. </li><li> Orta ve kültür% 5 CO2 ve başka bir 48 saat süre ile 37 ° C &#39;de iskeleleri yenile; Daha sonra, orta 200 U kollajenaz IV (100 U / ml) eklenerek iskele hücreleri bırakın. </li><li> Sonra yayımlanmış hücreleri aşağı doğru döndürün. B27, VEGF-A (50 ng / ml) ve SB-431542 (10 uM)% 2 ile takviye edilmiş 2 mi EGM2-OG ortamı orta yerine; iki günde orta yenileyin. </li><li> 100 U / ml kolajenaz IV hücreleri ayırmak yaklaşık 10 gün sonra (~ farklılaşma başlatıldıktan sonra 14. günde, yani, h) izoleAkış sitometrisi ile farklılaşmış hücre popülasyonundan iPSC-EC AT özel markör proteinlerin ekspresyonu için analiz (örneğin, CD31, CD144) 3. </li><li> Kurulmuş bir protokole 3 yoluyla izole hiPSC-ECS genişletin. </li></ol></li></ol> 3. hiPSC-SMC&#39;ler içine hiPSCs Farklılaşan <ol><li> Jelatinimsi protein karışımı ile kaplanmış plakalar üzerine hiPSCs tohum, kültür mTeSR1 ortamı içinde hücreler,% 5 CO2 ve 37 ° C&#39;de, gün ortam değişiklikleri ile, birbirine karışana kadar (yaklaşık 2 gün). </li><li> 3 gün RPMI1640 ortam maddesi ve% 2 B27 artı insülin CHIR99021 (5 uM) ve BMP-4 (10 ng / ml) ile hücreleri kültürleyerek mezodermal soyundan hücreler içine farklılaşmasını başlatın. </li><li> Ağırlıklı sentetik fenotip hiPSC-SMC&#39;ler üretmek için: <ol><li> Kültür, VEGF-A (25 ng / ml), RPMI1640 me fibroblast büyüme faktörü p (FGFβ, 5 ng / ml) ile hücrelerin<html> dium ve% 2 B27 eksi insülin,% 5 CO2 ve 4 gün boyunca 37 ° C. </li><li> Kültür, VEGF-A, hücreler (25 ng / ml), FGFβ% 5 CO2 ve 37 ° C&#39;de RPMI-1640 ortamı içinde (5 ng / ml) ve% 2 B27 artı insülin 2 gün ° C. </li><li> Kültür trombosit türevli büyüme faktörü p (PDGFβ, 10 ng / ml), TGFβ% 5 CO2 ve 37 ° RPMI1640 (3 ng / ml) ve% 2 B27 artı insülin hücreler 4 gün boyunca ° C. </li></ol></li><li> Ağırlıklı kasılma fenotip hiPSC-SMC&#39;ler üretmek için: <ol><li> Kültür 4 RPMI1640 VEGF-A ile günde (25 ng / ml) ve FGFβ (5 ng / ml) ve% 2 B27 eksi insülin hücreleri. </li><li> Kültür RPMI1640 ve% 2 B27 artı insülin PDGFβ (5 ng / ml) ve TGFβ (2.5 ng / ml) ile 7 gün hücreler. </li></ol></li><li> ~ 6 gün RPMI1640 metabolik ortam içeren laktat (4 mM) içinde kültürlenmesi ile son hiPSC-SMC popülasyonları arındırın. </li></ol>jove_title &quot;&gt; 4. IGF-içeren Mikroküreler oluşturma <ol><li> Isı Zeytin yağı bir su banyosu içinde 45 ° C arasındadır. </li><li> Isı 50 ° C&#39;ye% 10 jelatin çözeltisi 5 mi. </li><li> zeytin yağı, karışmaya jelatin ekleyin ve bir su banyosu buz ilave edilerek 5 ° C&#39;ye kadar hızlı bir şekilde soğutulur. </li><li> Yirmi beş dakika sonra, mikro küre oluşumunu uyarmak için, zeytin yağı soğutulmuş (4 ° C) aseton ilave edin. </li><li> 5 de sıcaklığını muhafaza 1 saat boyunca ° C; daha sonra, zeytin yağı çıkarmak için önceden soğutulmuş asetonla onları 5 kez yıkayın, mikro küreler toplamak ve 4 ° C&#39;de kurumaya izin verin. </li><li> çapraz bağlama uyarılması için gece boyunca 4 ° C &#39;de% 70 etanol ve% 0.25 glutaraldehid çözeltisi içinde küreler tekrar süspansiyon ve daha sonra 100 mM glisin Karışımı nötralize. </li><li> Saat 30 dakika boyunca 5 ug IGF ve% 0.1 sığır serum albümini ihtiva eden 2 O damıtılmış 15 ul 5 mg mikro-küreler karıştırılarak mikrosferler olarak yüklenebilir IGF. </li></ol> 5. Hücre Yaralanma Sitesi üzerinde yama oluşturma ve enjekte <ol><li> 1 ml Minimum Essential Medium (MEM) birlikte hiPSC kaynaklı SMC&#39;lere (1 milyon dolar) CMS (2 milyon), ve ECS (1 milyon dolar) Askıya. </li><li> 1 mi fibrinojen çözeltisi (25 mg / ml) içinde mikro-5 mg süspanse edin. </li><li> Hücre-içeren çözelti, fibrinojen / mikro küre çözeltisi ile bir şırınga ve trombin çözeltisinin 1 ml&#39;si ile üçüncü bir şırınga ile bir şırınga doldurun (80 NIH birim / mi, 2 ul 400 mM CaCl2, 200 mM ε-aminokaproik ile desteklenmiş asit). </li><li> Cerrahi 2 daha önce açıklandığı gibi inen koroner arter sol ön ligasyonu yoluyla domuz kalbinde miyokard enfarktüsü neden olur. NOT: Kısa, dişi Yorkshire domuz olarak (~ 13 kg, yaş 45 gün) Telazol / Xylazine (4.4 mg / kg) kas içi enjeksiyon ile sedasyon edilir ve izofluran (% 0.5-5) ile genel anestezi altında entübe. Bir 4. IntercOstal torakotomi ve maruz kaldığı inen koroner arter anterior bırakılır. koroner arter 60 dakika boyunca bağlanması ile tıkanır ve daha sonra bağ kaldırılır. Hemen elektriksel defibrilasyon ventriküler fibrilasyon durumunda gerçekleştirilir. </li><li> domuz kalplerin infarkt bölgenin epikarda bir steril plastik halka (~ 2.5 cm) yerleştirin ve sonra aynı anda ringe kürecik / fibrinojen ve trombin çözümleri enjekte edin. </li><li> Karışım (~ 30 sn) kuvvetlendirmek için izin ve sonra hücrelerin infarkt miyokard içine katılaşmış karışımın içinden hücre içeren şırınga iğne takın ve enjekte. 3-0 ya da 2-0 monocryl sütür hayvan boyutuna bağlı olarak kas katmanları, deri altı dokusu ve göğüs duvarı kapatın. Buprenorfin 0.01-0.02 mg / kg kas içi enjeksiyon her 8 saatte bir ameliyat sonrası ilk 3 gün ağrıyı kontrol etmek için kullanılır. </li><li> kardiyak manyetik rezonans kardiyak fonksiyonu değerlendirmekcerrahi işlem, bir hafta ve cerrahi işlem 2, 3, 4 sonraki dört hafta önce görüntüleme (MRG). Son MRG çalışmalarından sonra, hayvan kurban ve histoloji ve moleküler analiz 2, 3, 4 için kalpleri işlemek. </li></ol>

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hiPSC-CM&#39;ler gelişmiş Verim / Saflık CM&#39;ler içine insan kök hücreleri farklılaştırarak için geleneksel protokoller genellikle düşük verim ve saflık ile sınırlıdır; Örneğin, hESC CM&#39;ler sadece 35-66% yavaş miyozin ağır zincir ya da cTnT 6 dile Percoll ayrılması ve kardiyak vücut oluşumu yoluyla elde edilmiştir. Ayırt hiPSC-CM popülasyonlarının saflığı esasen promotere işlevsel olarak bağlanmış olan bir raportör geninin s...

onlar hastanın bağışıklık sistemi tarafından reddedilmesi olmayan hücrelerin bir potansiyel olarak sınırsız aralığı ve miktar içine ayırt edilebilir, çünkü insan uyarılmış pluripotent kök hücreler (hiPSCs) rejeneratif hücre tedavisi için en umut verici ajanlar arasında yer almaktadır. (SMC&#39;lere Bununla birlikte, kendi kendine replikasyon ve farklılaşması için kendi kapasitesi, tümör oluşumuna neden olabilir ve bunun sonucunda, hiPSCs tamamen bu kardiyomiyositlerde (CMS), endotel hücreleri (EC), ve düz kas hücreleri gibi özel hücre tipleri, ayırt edilmesi gereken ), uygulamadan önce. Hücre yönetiminin en basit ve en yaygın yöntemlerden biri direkt intramiyokardiyal enjeksiyon, ancak yerli miyokard dokusu ile aşılanan nakledilen hücrelerin sayısı son derece azdır. Bu yıpratma çok iskemik doku sitotoksik ortamına bağlanabilir; Ancak, fare embriyonik kök hücreler (EKH) iken yaralanmamış kalplerin, myokarddan içine doğrudan enjekte ouygulanan hücrelerin önemli bir bölümünün onlar esnasında üretilen yüksek basınçlar tarafından iğne izi aracılığıyla sıkılmış, belki de yönetim sitesine çıkıldı düşündürmektedir 3-5 saat 1 için tutuldu teslim 5 milyon hücre nly ~ 40% miyokard kasılma. Burada, hiPSC türetilmiş kardiyomiyositlerde (hiPSC CMS) 2, endotel hücrelerinin (hiPSC-EC) 3 ve düz kas hücreleri (SMC&#39;lere) 4 oluşturmak için yeni ve büyük ölçüde daha etkili yöntemler sunar. Özellikle, bu hiPSC-SMC protokol ağırlıklı sentetik veya kontraktil SMC fenotip doğru hücreleri yönlendirerek somatik DKH&#39;lerde 5 gözlenen morfolojik ve fonksiyonel özelliklerinin geniş taklit etmek ilk. Ayrıca, bir sitokin içeren fibrin s oluşturarak enjekte edilen hücrelerin melezleşmesi hızını artırır, hücre dağıtım için bir yöntem sağlarEnjeksiyon sitesi üzerinden atch. Yama, en az üç günlük bir süre boyunca insülin benzeri büyüme faktörü (IGF) serbest bırakarak, miyokard çıkan hücreler önlemek için iğne parça, ve hücre yaşamını mühürlenerek, her iki hücre tutmanın geliştirilmesi görünmektedir.

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Protocol 1</td>
			<td style="width:180px;"></td>
			<td style="width:155px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 medium</td>
			<td>Stem cell technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth-factor-reduced matrigel</td>
			<td>Corning lifescience</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Stem cell technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement, serum free</td>
			<td>Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BMP-4</td>
			<td>R&#38;D</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bFGF</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase IV</td>
			<td>Fisher Scientific</td>
			<td>NC0217889</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:163px;">Hanks Balanced Salt Solution (Dextrose, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 anhydrous)</td>
			<td>Fisher Scientific</td>
			<td>14175079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10438018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate</td>
			<td>Corning Lifescience</td>
			<td>356721</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm dish</td>
			<td>Corning Lifescience</td>
			<td>354732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene</td>
			<td>Fisher Scientific</td>
			<td>15040066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibrinogen</td>
			<td>Sigma-Aldrich</td>
			<td>F8630-5g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T7009-1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMB2 medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VEGF</td>
			<td>ProSpec-Tany</td>
			<td>CYT-241</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EPO</td>
			<td>Life Technologies</td>
			<td>PHC9431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TGF-&#223;</td>
			<td>Peprotech</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGM2-MV medium</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB-431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD31</td>
			<td>BD Bioscience</td>
			<td>BDB555445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD144</td>
			<td>BD Bioscience</td>
			<td>560411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>12565269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorff Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR99021</td>
			<td>Stem cell technologies</td>
			<td>720542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDGF-&#223;</td>
			<td>Prospec</td>
			<td>CYT-501-10ug</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olive oil</td>
			<td>Sigma-Aldrich</td>
			<td>O1514</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGF</td>
			<td>R&#38;D</td>
			<td>291-G1-01M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>15561020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heating plate</td>
			<td>Fisher Scientific</td>
			<td>SP88850200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"><img alt="ezh" src="/files/ftp_upload/55142/ezh.jpg" />-aminocaproic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A0420000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM medium</td>
			<td>Fisher Scientific</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>Fisher Scientific</td>
			<td>1482748</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Datex-Ohmeda</td>
			<td>47810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Ohio Medical</td>
			<td>V5A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defibrillator</td>
			<td>Physiol Control</td>
			<td>LIFEPAK 15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5T MRI</td>
			<td>General Electric</td>
			<td>Signa Horizon LX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7T MRI</td>
			<td>Siemens</td>
			<td>10018532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gadolinium Contrast Medium (Magnevist)</td>
			<td>Berlex</td>
			<td>50419-188-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-0 silk suture</td>
			<td>Ethilon</td>
			<td>685H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 silk suture</td>
			<td>Ethilon</td>
			<td>622H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 monofilament suture</td>
			<td>Ethilon</td>
			<td>627H</td>
			<td></td>
		</tr>
	</tbody>
</table>

pluripotent stem cell derived cardiac cells for myocardial repair

İnsan uyarılmış pluripotent kök hücrelerin (hiPSCs) tamamen kardiyomiyositlerde (hiPSC-CMS), endotel hücreleri (hiPSC-EC), ve düz kas hücreleri (SMC&#39;lere) içine hiPSCs ayrıştırılması için uygulamadan önce, özel hücre tipleri, fakat geleneksel protokolleri ayırt edilmelidir genellikle düşük verim, saflık ve / veya zayıf fenotipik stabilitesi ile sınırlıdır. Burada, yeni hiPSC CMS, -ECs üretilmesi için protokoller ve -SMCs esas itibarıyla geleneksel yöntemlere göre daha verimli, hem de uygulama alanı üzerine oluşturulan bir sitokin içerikli bir flaster ile hücre enjeksiyonu birleştirmek için bir yöntem sunulmaktadır. Yama uzun bir süre boyunca insülin benzeri büyüme faktörü (IGF) serbest bırakarak, miyokard sıkılmış olan hücreler önlemek için iğne parça, ve hücre yaşamını mühürlenerek, enjekte edilen hücrelerin tutma hem de arttırır. miyokardiyal iskemi-reperfüzyon hasarının domuz modelinde olarak bütünleşme oranı daha yüksek, iki kat daha fazla olduğunuHücreler hücreleri ve yama hem sitokin içeren yama olmadan hücrelere kıyasla yama ve tedavi ile uygulanmıştır, fakat tek başına hücreleri ile, kalp fonksiyonu ve infarkt önemli iyileşmeler ile ilişkilidir.

Farklılaştırılmış hiPSC-CM&#39;ler, -ECs ve -SMCs karakterizasyonu HiPSCs ayırıcı kapasitesi, 4, 2 3 değerlendirildi. Sitometri son hiPSC-CM nüfusun saflık% 90 (Şekil 1A, 1B, panel B1) aşabilir düşündürmektedir kardiyak troponin T (cTnT) ifade analizleri Akış. Hemen hemen bütün hüc...

Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Biz kardiyomiyositlerde, endotel hücreleri ve düz kas hücreleri ve yama-aracılı sitokin teslimat hücre enjeksiyon birleştirerek nakledilen hücrelerin bütünleşmesini geliştiren bir dağıtım yöntemi, insan uyarılmış pluripotent kök hücrelerin ayırt etmek için, üç yeni ve daha etkin protokollere sunuyoruz.

Pluripotent Kök Hücre Miyokard Onarım Kardiyak Hücreleri Türetilmiş

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

pluripotent-stem-cell-derived-cardiac-cells-for-myocardial-repair

Research

JoVE Journal

Developmental Biology

8.2K Görüntüleme.  University of Alabama at Birmingham.  Biz kardiyomiyositlerde, endotel hücreleri ve düz kas hücreleri ve yama-aracılı sitokin teslimat hücre enjeksiyon birleştirerek nakledilen hücrelerin bütünleşmesini geliştiren bir dağıtım yöntemi, insan uyarılmış pluripotent kök hücrelerin ayırt etmek için, üç yeni ve daha etkin protokollere sunuyoruz. 

Video: Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Myh6-Promotörle Seçim ile kombinasyonu ES-Hücre-Programlama dayanarak Kemirgen Kardiyak Kalp Pili Hücre Agregaların Üretimi

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

Gelişim Biyolojisi

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Saç türetilmiş keratinositlerden Kullanarak Entegrasyon serbest İnsan uyarılmış pluripotent kök hücrelerin üretilmesi

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

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.

Insan pluripotent kök hücreleri gelen melanosit besleyici içermeyen türetilmesi

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

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Oldukça Tekrarlanabilir Küçük Molekül Tabanlı Farklılaşma Protokolü kullanarak insan pluripotent kök hücreleri gelen kardiyomiyositlerde Büyük Ölçekli Üretim

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Bir Transpozon Sistemi Kullanılarak Fare Amniyotik Sıvı Hücreleri pluripotent kök hücrelerin üretimi

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Kordon Kanından Oluşan İndüklü Pluripotent Kök Hücrelerden Kondrojenik Pellet Oluşumu

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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.

Hızlı, yönlendirilmiş farklılaşma insan embriyonik veya İndüklenmiş Pluripotent kök hücrelerinden retina Pigment epitel hücrelerinin

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.

Yaşam sağlam Zebra balığı tek hücreli Photoconversion

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 ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

İnsan kullanarak Pluripotent kök hücre kaynaklı hepatosit benzeri hücreler indüklenen ilaç bulma için

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

İndüklenmiş Pluripotent kök hücre kaynaklı tetkikine kullanarak ailesel hiperkolesterolemi insan karaciğer Chimeric fare modeli

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...