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).

Alle experimentellen Verfahren werden in Übereinstimmung mit den Tier Richtlinien der University of Alabama in Birmingham durchgeführt. 1. Differenziert hiPSCs in hiPSC-CMs <ol><li> Beschichten der Vertiefungen einer Platte mit 6 Vertiefungen mit vorgekühlten wachstumsfaktor reduziert gelatinösen Proteingemisch bei 4 ° C über Nacht. Saugen Sie das gallertartig Proteinmischung vor dem Gebrauch. Seed die hiPSCs auf die vorbeschichteten Platten und Kultur der Zellen (1 × 10 5 Zellen pro Vertiefung) bei 5% CO 2 und 37 ° C in mTeSR1 Medium Ergänzung mit 10 uM ROCK - Inhibitor. </li><li> Aktualisieren Sie das Medium täglich, bis die Zellen 90% Konfluenz erreichen; dann, wachstumsfaktor reduziert gelatinösen Proteinmischung zu dem Medium (0,5 mg gelatinösen Proteinmischung pro Platte mit 6 Vertiefungen, 2 ml Medium pro Vertiefung), und die Kultur der Zellen für 5% CO 2 und 37 ° C zwei Tage hinzu. <ol><li> Um das Medium zu ersetzen, saugen sanft das Medium aus der Petrischale über Vakuum mitaus den Zellen zu berühren, und neues Medium mit einer Transferpipette hinzufügen. </li></ol></li><li> Initiieren Differenzierung durch das Medium mit RPMI1640-Medium ersetzt, supplementiert mit Wachstumsfaktor reduzierte gelatinösen Proteingemisch, B27 ohne Insulin und 100 ng / ml Activin A; Kultur die Zellen bei 5% CO 2 und 37 ° C für 24 Std. </li><li> Ersetzen des Mediums mit RPMI1640-Medium, das ohne Insulin mit B27 ergänzt wurde, 10 ng / ml bone morphogenic protein 4 (BMP-4) und 10 ng / ml basischen Fibroblastenwachstumsfaktor (bFGF); Kultur die Zellen bei 5% CO 2 und 37 ° C für 96 Stunden. </li><li> Ersetzen Sie das Medium mit RPMI1640 - Medium mit B27 ergänzt und weiter Kultur der Zellen bei 5% CO 2 und 37 ° C; erfrischen alle 3 Tage das Medium. </li><li> Beobachten Cluster von Zellen unter dem Mikroskop ~ 3 Tage nach Beginn der Differenzierung zieht. Sammeln Sie die Cluster ~ 7 Tage nach der ersten Kontraktionen und waschen Sie sie in Hanks Balanced Salt Solution Beobachtung. <ol><li> Distanzieren der geclusterten-Zellen in der Platte, indem sie in Hanks-Puffer Inkubation für 10 min bei 37 ° C, die 100 U / ml Kollagenase IV unter leichtem Schütteln; Dann wird für 5 min 0,25% Trypsin in Ethylendiamintetraessigsäure (EDTA) hinzuzufügen. </li><li> Neutralisieren der Enzymlösung mit 10% fötalem Rinderserum in RPMI / B27-Medium und dann Resuspendieren der Zellen in RPMI / B27 Medium. </li><li> Zähle die Anzahl der Zellen durch Hämocytometer. Kultur die Zellen auf 10 cm - Schalen bei 5% CO 2 und 37 ° C für mindestens 3 Stunden, die die nicht-Kardiomyozyten - Zellen an die Oberfläche der Kulturschale anhaften können. </li></ol></li><li> Sammeln Sie die Zellsuspension, die die hiPSC-CMs enthält, und halten Sie die Zellen (etwa 1 x 10 6 Zellen pro 10 cm Schale) , indem sie auf einer gallertartigen Proteinmischung -beschichteten Oberfläche kultiviert werden . </li></ol> 2. Differenziert hiPSCs in hiPSC-ECs <ol><li> Distanzieren die hiPSC Bevölkerung in einzelne Zellen durch Inkubation them mit Mittel bei 5% CO 2 und 37 ° C für 5 min chelatisierenden. Übertragen Sie die Zellen in ein 15 ml Zentrifugenröhrchen mit frischem mTeSR1 Medium. </li><li> Spin down die Zellen bei 200 xg für 5 min. Resuspendieren der Zellen in mTeSR1 Medium, das mit 10 &amp; mgr; M ROCK-Inhibitor. Bestimmen Sie die Zelldichte mit einem Hämozytometer. </li><li> In 250 ul 20 NIH-Einheiten / ml Thrombin-Lösung auf eine Vertiefung einer 24-Well-Platte. </li><li> 1 x 10 6 hiPSCs bis 250 &amp; mgr; l einer 12,5 mg / ml Fibrinogen - Lösung. Und fügen Sie die 250 ul zellhaltigen Fibrinogenlösung auf die Thrombin-geladen gut. Beachten Sie die Mischung verfestigen, um ein hiPSC haltigen Fibrin Gerüst innerhalb min zu bilden. </li><li> Übertragen die verfestigten, zellhaltigen Gerüste in die Vertiefungen einer Platte mit 6 Vertiefungen mit den Deckglaspinzetten und Differenzierung einzuleiten durch Kultivieren der Gerüste in 2 ml EBM2 Medium, ergänzt mit 2% B27 ohne Insulin, Activin-A (50 ng / ml) und BMP-4 (25 ng / ml) für 24 h bei 5% CO <sub&gt; 2 und 37 ° C. <ol><li> Ersetzen Sie das Medium vorsichtig das alte Medium über Vakuum saugt, ohne die Zellen zu berühren. Hinzufügen neuer EBM2 Medium, supplementiert mit B27 ohne Insulin, vaskulären endothelialen Wachstumsfaktor (VEGF, 50 ng / ml), Erythropoietin (EPO, 100 ng / ml) und transformierender Wachstumsfaktor β1 (TGFβ1, 10 ng / ml), und die Kultur, die Gerüste bei 5% CO 2 und 37 ° C für 48 Stunden. </li><li> Aktualisieren Sie die mittlere und Kultur die Gerüste bei 5% CO 2 und 37 ° C für weitere 48 h; Dann lassen Sie die Zellen aus dem Gerüst durch Zugabe von 200 U Kollagenase IV (100 U / ml) zum Medium. </li><li> Spin down die Zellen, nachdem sie freigegeben werden. Ersetzen Sie das Medium mit 2 ml EGM2-MV-Medium mit 2% der B27 ergänzt, VEGF-A (50 ng / ml) und SB-431542 (10 &amp; mgr; M); aktualisieren alle zwei Tage das Medium. </li><li> Etwa 10 Tage später (dh auf ~ 14. Tag nach der Differenzierung initiiert wurde), distanzieren die Zellen mit 100 U / ml Kollagenase IV, isolieren hiPS-ECs aus der Population von differenzierten Zellen über Durchflusszytometrie analysiert für die Expression von EC-spezifischen Markerproteine (zB CD31, CD144) 3. </li><li> Erweitern Sie den isolierten hiPSC-ECs über ein etabliertes Protokoll 3. </li></ol></li></ol> 3. Differenziert hiPSCs in hiPSC-SMCs <ol><li> Samen der hiPSCs auf Platten, die die Zellen in mTeSR1 Medium bei 5% CO 2 und 37 ° C, mit täglicher Mediumwechsel, bis zur Konfluenz (~ 2 Tage) mit gelatinösen Proteingemisch, und die Kultur beschichtet wurden. </li><li> Initiieren Differenzierung in mesodermalen Abstammungslinien-Zellen durch Kultivieren der Zellen mit CHIR99021 (5 uM) und BMP-4 (10 ng / ml) in RPMI1640-Medium und 2% B27 plus Insulin für 3 Tage. </li><li> HiPSC-SMCs mit überwiegend synthetischen Phänotyp zu erzeugen: <ol><li> Kultur der Zellen mit VEGF-A (25 ng / ml), Fibroblasten-Wachstumsfaktor-β (FGFβ, 5 ng / ml) in RPMI1640 me<html> dium und 2% B27 minus Insulins bei 5% CO 2 und 37 ° C für 4 Tage. </li><li> Kultur der Zellen in VEGF-A (25 ng / ml), FGFβ (5 ng / ml) in RPMI1640 - Medium und 2% B27 plus Insulin bei 5% CO 2 und 37 ° C für 2 Tage. </li><li> Kultur der Zellen in platelet-derived growth factor β (PDGFβ, 10 ng / ml), TGF &amp; bgr; (3 ng / ml) in RPMI - 1640 und 2% B27 plus Insulin bei 5% CO 2 und 37 ° C für 4 Tage. </li></ol></li><li> HiPSC-SMCs mit überwiegend Kontraktions Phänotyp zu erzeugen: <ol><li> Kultur die Zellen für 4 Tage mit VEGF-A (25 ng / ml) und FGFβ (5 ng / ml) in RPMI-1640 und 2% B27 minus Insulin. </li><li> Kultur die Zellen für 7 Tage mit PDGFβ (5 ng / ml) und TGF &amp; bgr; (2,5 ng / ml) in RPMI-1640 und 2% B27 plus Insulin. </li></ol></li><li> Reinige den endgültigen hiPSC-SMC-Populationen, indem die Zellen in Lactat-Kultivierung (4 mM) RPMI1640 Stoffwechselmedium für ~ 6 Tage enthält. </li></ol>jove_title &quot;&gt; 4. Erstellen der IGF-enthaltenden Microspheres <ol><li> Olivenöl erhitzen auf 45 ° C in einem Wasserbad. </li><li> Wärme 5 ml 10% igen Gelatinelösung auf 50 ° C. </li><li> Fügen Sie die Gelatine zu dem Olivenöl, rühren, und dann schnell abkühlen bis 5 ° C durch das Eis in das Wasserbad Zugabe. </li><li> Fünfundzwanzig Minuten später hinzuzufügen gekühlt (4 ° C) Aceton zu dem Olivenöl Mikrokügelchens Bildung zu induzieren. </li><li> Man hält die Temperatur bei 5 ° C für 1 Stunde; Dann sammeln Sie die Microspheres, sie 5-mal mit vorgekühltem Aceton waschen das Olivenöl, und lassen Sie sie an der Luft trocknen bei 4 ° C zu entfernen. </li><li> Resuspendieren der Mikrokügelchen in einer Lösung aus 70% Ethanol und 0,25% Glutaraldehyd über Nacht bei 4 ° C eine Vernetzung zu induzieren, und dann die Mischung mit 100 mM Glycin zu neutralisieren. </li><li> Last IGF in die Mikrokügelchen durch Mischen von 5 mg Mikrosphären mit 15 &amp; mgr; l destilliertem H 2 O , enthaltend 5 &amp; mgr; g IGF und 0,1% Rinderserumalbumin für 30 min. </li></ol> 5. Erstellen der Patch über der Stelle der Verletzung und die Injektion der Zellen <ol><li> Hängen Sie den hiPSC abgeleiteten CMs (2 Millionen), SMCs (1 Million) und ECs (1 Million) zusammen in 1 ml Minimum Essential Medium (MEM). </li><li> Suspend 5 mg Mikrokügelchen in 1 ml Fibrinogen-Lösung (25 mg / ml). </li><li> Füllen einer Spritze mit der zellhaltigen Lösung, eine andere Spritze mit der Fibrinogen / Mikrokügelchen - Lösung und eine dritte Spritze mit 1 ml Thrombinlösung (80 NIH Einheiten / ml, ergänzt mit 2 &amp; mgr; l 400 mM CaCl 2 und 200 mM ε-Aminocapronsäure Acid). </li><li> Chirurgisch Myokardinfarkt bei Schweinen Herz über Ligatur der linken vorderen absteigenden Koronararterie induzieren , wie vor 2 beschrieben. HINWEIS: Kurz gesagt, weibliche Yorkshire Schweine (~ 13 kg, 45 Tage alt) mit intramuskuläre Injektion von Telazol / Xylazin sediert sind (4,4 mg / kg) und unter Vollnarkose mit Isofluran (0,5-5%) intubiert. A 4 th intercostal Thorakotomie Koronararterie vorderen absteigenden ausgeführt und links ausgesetzt ist. Die Koronararterie wird durch eine Ligatur für 60 min okkludiert und dann wird die Ligatur entfernt. Sofortige elektrische Defibrillation wird im Fall von ventrikulärer Fibrillation durchgeführt. </li><li> Positionieren Sie einen sterilen Kunststoffring (~ 2,5 cm) auf dem Epikard des Infarktregion Schweineherzen, und dann zu injizieren gleichzeitig die Mikrokügelchen / Fibrinogen und Thrombin-Lösungen in den Ring. </li><li> Lassen Sie die Mischung (~ 30 sec) zu verfestigen, und dann legen Sie die Nadel der zellhaltigen Spritze durch die verfestigte Mischung in das Infarkt Myokard und injizieren die Zellen. Schließen Sie die Muskelschichten, Unterhautgewebe, und der Brustwand mit 3-0 oder 2-0 Monocryl Naht je nach Tiergröße. Buprenorphin 0,01-0,02 mg / kg intramuskulär alle 8 Stunden verwendet werden, um Schmerzen für die ersten 3 Tage nach der Operation zu steuern. </li><li> Bewerten Sie die Herzfunktion eines Herz mittels magnetischer Resonanzimaging (MRI) vor dem chirurgischen Eingriff, einer Woche und vier Wochen nach der chirurgischen Prozedur 2, 3, 4. Nach dem abschließenden MRT - Studien, opfern , um das Tier und verarbeiten ihre Herzen für die Histologie und molekulare Analyse 2, 3, 4. </li></ol>

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Verbesserte Ausbeute / Reinheit von hiPSC-CMs Herkömmliche Protokolle für die oft menschliche Stammzellen in CMs Differenzierung durch eine geringe Ausbeute und Reinheit begrenzt; zum Beispiel erhalten nur 35-66% der hESC-CMs über Percoll Trennung und Herzkörperbildung langsam Myosin schwere Kette oder cTnT 6 ausgedrückt. Die Reinheit von differenzierten hiPSC-CM - Populationen im wesentlichen durch die Auswahl für die Expression eines Reportergens erhöht werden, ...

Menschliche induzierte pluripotente Stammzellen (hiPSCs) gehören zu den vielversprechendsten Mittel für die regenerative Zelltherapie, weil sie in eine potenziell unbegrenzte Reichweite und Menge der Zellen unterschieden werden können, die durch den Patienten Immunsystem abgestoßen werden nicht. Aber ihre Fähigkeit zur Selbstreplikation und die Differenzierung kann auch zur Tumorbildung führen und folglich müssen hiPSCs vollständig in spezifische Zelltypen zu differenzieren, wie Kardiomyozyten (CMs), Endothelzellen (ECs) und glatten Muskelzellen (SMCs ), vor der Verabreichung. Eine der einfachsten und häufigsten Methoden der Zell Verabreichung direkte intramyokardiale Injektion, aber die Anzahl der transplantierten Zellen, die von der nativen Herzmuskelgewebe transplantiert werden, ist außerordentlich gering. Ein Großteil dieser Abrieb kann auf die zytotoxische Umgebung des ischämischen Gewebes zugeschrieben werden; Wenn jedoch murinen embryonalen Stammzellen (ES-Zellen) wurden direkt in das Myokard von unversehrten Herzen injiziert, only ~ 40% der 5 Millionen Zellen wurden für 3-5 h 1, behielt geliefert , die ein wesentlicher Teil der verabreichten Zellen legt nahe , dass die Applikationsstelle verlassen, vielleicht , weil sie durch die hohen Drücke durch die Nadelbahn herausgedrückt erzeugt während Myokardkontraktion. Hier stellen wir neuartige und wesentlich effizientere Methoden zur Erzeugung hiPSC abgeleitete Kardiomyozyten (hiPSC-CMs) 2, Endothelzellen (hiPSC-ECs) 3 und glatten Muskelzellen (SMCs) 4. Bemerkenswert ist , ist dies hiPSC-SMC - Protokoll die ersten , die breite Palette von morphologischen und funktionellen Eigenschaften in somatischen SMCs 5 beobachtet zu imitieren , indem die Zellen in Richtung einer überwiegend synthetische oder Kontraktions SMC - Phänotyp zu lenken. Wir stellen auch ein Verfahren zur Zellabgabe, die durch die Schaffung eines Cytokin-enthaltenden Fibrin p die Transplantationsrate von injizierten Zellen verbessertAtch auf die Injektionsstelle. Der Patch erscheint beide Zellretention zu verbessern, indem der Nadelbahn Abdichten der Zellen aus dem Verlassen des Myokards, und das Überleben der Zelle, durch die Freigabe insulin-like growth factor (IGF) über einen Zeitraum von mindestens drei Tage zu verhindern.

<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

Menschlichen pluripotenten Stammzellen (hiPSCs) müssen vollständig in spezifische Zelltypen vor der Verabreichung unterschieden werden, aber herkömmliche Protokolle für hiPSCs in Kardiomyozyten (hiPSC-CMs), Endothelzellen (hiPSC-ECs) und glatten Muskelzellen (SMCs) Differenzieren sind oft begrenzt durch geringe Ausbeute, Reinheit und / oder schlechter phänotypische Stabilität. Hier stellen wir neue Protokolle zum Erzeugen hiPSC-CMs, -ECs und -SMCs, die wesentlich effizienter als herkömmliche Verfahren sind, sowie ein Verfahren zum Kombinieren von Zellinjektion mit einem Cytokin Haltiges Pflaster über der Verabreichungsstelle geschaffen. aus dem Myokard gequetscht, und das Überleben der Zelle, durch die Freigabe insulin-like growth factor (IGF) über einen längeren Zeitraum der Patch verbessert sowohl die Beibehaltung der injizierten Zellen, indem die Nadelbahn Abdichten der Zellen aus zu verhindern. In einem Schweinemodell der myokardialen Ischämie-Reperfusionsverletzung, war die Rate der Verpflanzung mehr als das Zweifache größer, wenn dieZellen wurden mit dem Zytokin Haltiges Pflaster zu den Zellen ohne Patch vergleicht verabreicht und die Behandlung sowohl mit den Zellen und dem Patch, aber nicht mit den Allein-Zellen wurde mit signifikanten Verbesserungen in der Herzfunktion und die Infarktgröße zugeordnet ist.

Charakterisierung von Differentiated hiPSC-CMs, -ECs und -SMCs Die differentielle Kapazität von hiPSCs 2 wurden ausgewertet, 3, 4. Durchflusszytometrie analysiert des kardialen Troponin T (cTnT) Ausdruck deuten darauf hin , dass die Reinheit des endgültigen hiPSC-CM Bevölkerung 90% überschreiten kann (Abbildung 1A, <...

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

Wir präsentieren drei neue und effizientere Protokolle für den menschlichen induzierten pluripotenten Stammzellen in Kardiomyozyten, Endothelzellen differenzieren und glatten Muskelzellen und eine Fördermethode, die das Einwachsen von transplantierten Zellen verbessert durch Zellinjektion mit Patch-vermittelten Cytokin-Lieferung zu kombinieren.

Pluripotente Stammzellen Herzzellen für Myocardial Repair Abgeleitet

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 Aufrufe.  University of Alabama at Birmingham.  Wir präsentieren drei neue und effizientere Protokolle für den menschlichen induzierten pluripotenten Stammzellen in Kardiomyozyten, Endothelzellen differenzieren und glatten Muskelzellen und eine Fördermethode, die das Einwachsen von transplantierten Zellen verbessert durch Zellinjektion mit Patch-vermittelten Cytokin-Lieferung zu kombinieren. 

Serie: 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.

Ableitung von Herzvorläuferzellen aus embryonalen Stammzellen

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

Forschung

Entwicklungsbiologie

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.

Generation Ausgerichtet Functional Myokardgewebe Durch Mikrokontaktdrucken

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

Biotechnik

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.

Beurteilung Stem Cell DNA-Integrität für kardiale Zelltherapie

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.

In-vitro- Differenzierung humaner mesenchymaler Stammzellen in funktionellen Cardiomyocyte-ähnlichen Zellen

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.

Unterdrückung der Pro-fibrotische Signalisierung potenziert Faktor-vermittelten Reprogrammierung von Maus embryonalen Fibroblasten in induzierte Kardiomyozyten

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.

Intramyokardiale Zell Lieferung: Beobachtungen im Herzen Murine

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

Medizin

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.

Induktion der endothelialen Differenzierung in kardialen Vorläuferzellen unter niedrigen Serum-Bedingungen

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.

Erzeugung und Expansion menschlicher Kardiomyozyten aus mononukleären Zellen des patientenperipheren Blutes

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.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit 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 ...

Biologie

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.

High Efficiency Differenzierung von humanen pluripotenten Stammzellen zu Kardiomyozyten und Charakterisierung mittels Durchflusszytometrie

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 ...