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><tbody><tr><td>&lt;strong&gt;Protocol Section 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>mTeSR1 medium</td><td>Stem cell technologies</td><td>5850</td><td></td></tr><tr><td>Growth-factor-reduced matrigel</td><td>Corning lifescience</td><td>356231</td><td></td></tr><tr><td>Y-27632</td><td>Stem cell technologies</td><td>72304</td><td></td></tr><tr><td>B27 supplement, serum free</td><td>Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>RPMI1640</td><td>Fisher Scientific</td><td>11875-119</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC-010</td><td></td></tr><tr><td>BMP-4</td><td>R&amp;amp;D</td><td>314-BP-010</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>232-FA-025</td><td></td></tr><tr><td>Collagenase IV</td><td>Fisher Scientific</td><td>NC0217889</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (Dextrose, KCl, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, NaHCO&lt;sub&gt;3&lt;/sub&gt;, NaCl, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; anhydrous)</td><td>Fisher Scientific</td><td>14175079</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10438018</td><td></td></tr><tr><td>6-well plate</td><td>Corning Lifescience</td><td>356721</td><td></td></tr><tr><td>10 cm dish</td><td>Corning Lifescience</td><td>354732</td><td></td></tr><tr><td>Cell incubator</td><td>Panasonic</td><td>MCO-18AC</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Versene</td><td>Fisher Scientific</td><td>15040066</td><td></td></tr><tr><td>Fibrinogen</td><td>Sigma-Aldrich</td><td>F8630-5g</td><td></td></tr><tr><td>Thrombin</td><td>Sigma-Aldrich</td><td>T7009-1KU</td><td></td></tr><tr><td>EMB2 medium</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>VEGF</td><td>ProSpec-Tany</td><td>CYT-241</td><td></td></tr><tr><td>EPO</td><td>Life Technologies</td><td>PHC9431</td><td></td></tr><tr><td>TGF-&amp;#946;</td><td>Peprotech</td><td>100-21C</td><td></td></tr><tr><td>EGM2-MV medium</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>SB-431542</td><td>Selleckchem</td><td>S1067</td><td></td></tr><tr><td>CD31</td><td>BD Bioscience</td><td>BDB555445</td><td></td></tr><tr><td>CD144</td><td>BD Bioscience</td><td>560411</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>Fisher Scientific</td><td>12565269</td><td></td></tr><tr><td>Eppendorff Centrifuge</td><td>Eppendorf</td><td>5702R</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CHIR99021</td><td>Stem cell technologies</td><td>720542</td><td></td></tr><tr><td>PDGF-&amp;#946;</td><td>Prospec</td><td>CYT-501-10ug</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 4&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Olive oil</td><td>Sigma-Aldrich</td><td>O1514</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818100</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898</td><td></td></tr><tr><td>IGF</td><td>R&amp;amp;D</td><td>291-G1-01M</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Fisher Scientific</td><td>15561020</td><td></td></tr><tr><td>Heating plate</td><td>Fisher Scientific</td><td>SP88850200</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>15-462-10Q</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 5&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>&amp;#949;-aminocaproic acid</td><td>Sigma-Aldrich</td><td>A0420000</td><td></td></tr><tr><td>MEM medium</td><td>Fisher Scientific</td><td>12561-056</td><td></td></tr><tr><td>Syringe</td><td>Fisher Scientific</td><td>1482748</td><td></td></tr><tr><td>Anesthesia ventilator</td><td>Datex-Ohmeda</td><td>47810</td><td></td></tr><tr><td>Anesthesia ventilator</td><td>Ohio Medical</td><td>V5A</td><td></td></tr><tr><td>Defibrillator</td><td>Physiol Control</td><td>LIFEPAK 15</td><td></td></tr><tr><td>1.5 T MRI</td><td>General Electric</td><td>Signa Horizon LX</td><td></td></tr><tr><td>7 T MRI</td><td>Siemens</td><td>10018532</td><td></td></tr><tr><td>Gadolinium Contrast Medium (Magnevist)</td><td>Berlex</td><td>50419-188-02</td><td></td></tr><tr><td>2-0 silk suture</td><td>Ethilon</td><td>685H</td><td></td></tr><tr><td>3-0 silk suture</td><td>Ethilon</td><td>622H</td><td></td></tr><tr><td>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

Universidad de Cartagena UDEC

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

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.

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 ...

Gelişim Biyolojisi

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Mikrokontak baskı sayesinde Bağlantısızlar Fonksiyonel Miyokard Doku Üretimi

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Biyomühendislik

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 ...

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

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Pro-fibrotik sinyal bastırma faktörü-aracılı fare embriyonik fibroblastlar indüklenen Cardiomyocytes yeniden programlama Potentiates

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

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Intramiyokardiyal Hücre Teslimat: Mürin Hearts Gözlemler

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Tıp

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 ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

Hasta Periferik Kan Mononükleer Hücrelerinden İnsan Kardiyomiyositlerinin Üretimi ve Genişlemesi

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

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 ...

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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 ...