Development of this protocol was supported by the Advancing a Healthier Wisconsin and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

HINWEIS: Die Patienten und / oder ihrer Erziehungsberechtigten sollten Einwilligung in einem Institutional Review Board teilzunehmen geben genehmigten Studie. 1. Puffer und Medien Vorbereitungen <ol><li> Waschpuffer: Zu 100 ml Waschpuffer vorbereiten, 1 ml der 100x Pen / Strep-Lösung (100 U / ml Penicillin + 100 ug / ml Streptomycin) und 99 ml phosphatgepufferter Kochsalzlösung (PBS). </li><li> Urinary Vorläuferzellen Cell (UPC) Medium: Um UPC Medium herzustellen, mischen gleichen Volumina von Keratinozyten Serum Free (KSF) Medium + Vorläuferzellen Cell Medium. <ol><li> KSF Medium: Um Keratinozytenmedium vorzubereiten, fügen Sie 5 ng / ml des epidermalen Wachstumsfaktors (EGF), 50 ng / ml Rinder (BPE), 30 ng / ml von Choleratoxin und Pen / Strep-Lösung (100 U / ml Penicillin, 100 ug / ml Streptomycin) versetzt, um 500 ml Medium KSF. </li><li> Vorläuferzellen Cell Medium: In drei Viertel der DMEM mit einem Viertel der Ham-F12-Medium und ergänzt mit 10% FBS, 0,4 ug / ml Hydrocortison, 0,1nM Choleratoxin, 5 ng / ml Insulin, 1,8 x 10 -4 M Adenin, 5 ug / ml Transferrin, 2 x 10 -9 M triiod Thyronin, 10 ng / ml EGF und Pen / Strep-Lösung. </li></ol></li><li> hES Medium: Fügen Sie 20% KO-Serumersatz (K-SR), 1x MEM nicht essentiellen Aminosäuren, 2 mM L-Glutamin, 100 uM β-Mercaptoethanol, 20 ng / ml bFGF und Pen / Strep zu 400 ml DMEM / F12-Medium. </li></ol> 2. Urinprobenentnahme <ol><li> Weisen Sie die Patienten, um Flüssigkeiten 30 Minuten vor dem Sammelurin trinken, um sicherzustellen, dass eine ausreichende Menge (~ 30-40 ml) der Urin gesammelt werden können. </li><li> Geben Sie dem Patienten und / oder deren Erziehungsberechtigte eine Urinsammlung Kit mit folgendem Inhalt: (1) schriftliche Anweisungen zur Steril- oder Reinfangurinprobe zu erhalten, (2) feuchte antibakterielle Toiletten, und (3) eine 100 ml sterile Proben-Sammelbecher. Patienten sollen angewiesen Probe zu sammeln. </li><li> Sofort legen Sie die Urinproben auf Eis und Transfer zum laboratory in einem Kühler. Verarbeiten Sie die Urin zur Zellisolierung sofort. Wenn eine Verzögerung unvermeidlich, lagern die Probe auf Eis für bis zu 4 h bei minimalem Verlust der Lebensfähigkeit der Zellen. </li></ol> 3. Isolierung und Expansion von Urin Cells HINWEIS: Führen Sie die folgenden Schritte unter sterilen Bedingungen in einem BSL2 Biologische Sicherheitswerkbank. <ol><li> Mit sterilen Pipetten, um den Transfer Urinproben 50-ml-Zentrifugenröhrchen steril. Zentrifuge bei 400 × g für 10 min bei RT. </li><li> Saugen Sie den Überstand zu verlassen 1 ml in der Röhre; darauf achten, dass das Zellpellet zu stören. </li><li> Resuspendieren und verbinden die Pellets aus einer Vielzahl von Röhren in 7 ml Waschpuffer und wiederholen Zentrifugation bei 400 × g für 10 min bei RT. </li><li> Vorsichtig den Überstand aspirieren, so dass die Zellpellet und ~ 0,2 bis 0,5 ml Überstand. Resuspendieren in 2-3 ml Urin-Vorläuferzellen Cell (UPC) Medium und übertragen Sie die Suspension in 4-6 Vertiefungen einer unbeschichtete 24-Well-Platte. </li><li> Nach 72 Stunden, ergänzen die Kultur mit 0,5 ml frischem UPC Medium pro Vertiefung und ändern Medium alle 2-3 Tage nach der. Die Erythrozyten und Schuppenzellen, die nicht an der Platte zu befestigen müssen mit den Mediumwechsel entfernt werden. </li><li> Überwachen Kultur nach 4-6 Tagen. HINWEIS: Kleine 2-4 Zellkolonien beginnt zu erscheinen. Cells wird abgerundet oder verlängert, die Typ I oder Typ II Nierenepithelzellen (RE) Zellen jeweils 3 darzustellen. </li><li> Ändern Sie das Medium alle 2-3 Tage, da, sobald die Zellen erscheinen, werden sie einen kräftigen Wachstumsphase durchlaufen. </li><li> Sobald die Zellen Konfluenz von 80-90% zu erreichen, in der Umgebung von 9-15 Tagen nach der Plattierung zu dissoziieren und den Durchgang der Zellen (15,000-18,000 Zellen / cm 2) auf 2-4 Vertiefungen der Platte mit 24 Vertiefungen zur weiteren Expansion. Kennzeichnen Sie diese als Zellpassage 1 (P1). </li><li> Charakterisieren Urinzellen für Linie Spezifikationen durch Durchflusszytometrie, immunohistochemchemische Färbung oder durch Reverse-Transkriptase-Polymerase-Kettenreaktion (RT-PCR). </li><li> Reprogram isoliert Urin Zellen (Abschnitt 4) oder frieren Sie sie in Freezing Medium (DMEM, ergänzt mit 10% Serum und 10% DMSO) für langfristige Kryolagerung. </li></ol> 4. Urin Zell Reprogrammierung Mit Sendai Virus HINWEIS: Der richtige Umgang mit und die Verwendung von PSA (persönliche Schutzausrüstung) wird empfohlen, während die Manipulation der Transfektion Mittel. Die folgenden Schritte werden unter sterilen Bedingungen in einem BSL2 Biologische Sicherheitswerkbank. Die ordnungsgemäße Entsorgung der Transfektion Mittel und / oder transfizierten Zellen wird empfohlen, um das Risiko von Umwelt- und Gesundheitsgefahren zu vermeiden. Für Urin Reprogrammierung, verwenden Sie ein Sendai Reprogrammierung Kit mit Änderungen an Feeder-abhängigen Protokoll des Herstellers wie unten beschrieben. <ol><li> Um einen hohen Wirkungsgrad Neuprogrammierung mit dem Sendai-Virus zu gewährleisten, verwenden Sie Gänge 1-5 von Urin-Zellen, die schnell sindly Division. Wenn die Zellen nicht gut replizieren oder werden seneszenten, verwerfen, da sie nicht effizient neu zu programmieren. </li><li> Seed 60.000 Zellen pro Vertiefung in zwei Vertiefungen einer Platte mit 6 Vertiefungen. Markieren Sie diese als Tag -2. Einzustellen Zellsaat Dichte (5 x Oktober 4-9 × 10 4 Zellen pro Vertiefung) wie erforderlich, um 80-90% Konfluenz von Tag 0 zu erreichen. </li><li> Am Tag 0 (48 h nach dem Ausplattieren der Zellen), bereiten die Sendai Umprogrammierung Vektoren (SeV), die jede der vier OSKM Faktoren. In jeder der Vektoren zu 1 ml vorgewärmten UPC Medium. Verwenden Sie eine MOI von 1-1,5 (5-7,5 x 10 5 CIU), die ausreicht, um eine Anpassung Urinzellen ist. Saugen Sie das Medium und langsam 1 ml UPC + SeV zu einer Vertiefung der Urin Zellen hinzuzufügen. Verwenden Sie die zweite sowie die Weiterleitung Negativkontrolle. </li><li> Am Tag 1, ersetzen Sie die UPC + SeV Medium durch frisches Medium UPC. Einige Zelltod nach 24 h durch virale Zytotoxizität gesehen. </li><li> Je nach Effizienz der umprogrammiert clonE-Formationen und die kompakte Morphologie, halten Wechsel des Mediums täglich bis Tag 6 oder 7. </li><li> Einen Tag vor der Dissoziation von transduzierten Zellen (Tag 5 oder 6), vorzubereiten MEF Nährplatten durch Plattieren Mitomycin-C (10 ug / ml für 3 Stunden) behandelt-MEFs bei einer Dichte von 5 x 10 4 Zellen / cm 2 auf 0,1 % Gelatine beschichtete 100 mm 2 Kulturschalen. </li><li> Am nächsten Tag (Tag 6 oder 7), dissoziieren die Zellen mit 0,25% Trypsin, Resuspendieren der Zellen in Medium und UPC Platte 5 × 10 4 - 2 x 10 5 Zellen / 100 mm 2 MEF Zuführplatte. </li><li> Am folgenden Tag zu hES Medium wechseln und das Medium jeden Tag. Beachten Sie die Zellen unter dem Mikroskop, um transformierte Zellen zu überwachen. HINWEIS: Die Zellen klonale Aggregaten mit typischen Kopfsteinpflastermorphologie und mit höheren Zellkern zu Plasma-Relation (12-18 Tage nach der Weiterleitung) zu bilden. </li><li> Innerhalb von zwei bis drei Wochen nach der Transduktion abholen Kolonien und Übertragung auf neue Platten für clonal Expansion. </li><li> Führen Lebendzellfärbung zum Auswählen iPSC Klone durch Inkubieren von Zellen mit TRA-1-81-Antikörper für 1 h bei 37 ° C im CO 2 -Inkubator nach Gegenfärbung mit spezifischen Fluoreszenzfarbstoff konjugierten sekundären Antikörper, gefolgt für 1 h bei 37 ° C im CO 2 -Inkubator. </li><li> Verwenden Sie eine 26 G 1½ Zoll Nadel Quer schlüpfen die größeren TRA-1-81 positive iPSC Klone in kleine gleichgroße Stücke (4-6 Stück pro Klon). </li><li> Verwenden Sie eine sterile Pipettenspitze zu holen und den Transfer 10-20 schraffierte Stücke aus mehreren Klonen in jede Vertiefung der Matrigel (10 ug / cm 2) beschichtete 24-Well-Platte mit hES-Medium. HINWEIS: Stücke von einem einzigen Klon einzeln in einer einzigen Vertiefung plattiert nicht erweitern oder pflegen Pluripotenz. </li><li> Ändern von hES zu iPSC Medium 24-48 Stunden nach der Umprogrammierung Klone werden auf die Matrigel Oberfläche befestigt. </li><li> Während der Wartung auf Matrigel-beschichteten Platten, manuell scrape-off alle differenzierten Zellen oder verunreinigen MEF Feeder-Zellen mit einer Pipette-Spitze bis zur voll reprogrammiert und pluripotent dystrophischen iPSC (MD-iPS) Klone anzureichern. </li><li> Nachdem einzelne Klone wurden ~ 100 &amp; mgr; m Größe erreicht, erweitern die Klone durch Dissoziieren mit 0,48 mM EDTA (Ethylendiamintetraessigsäure) Lösung für 3 min und Beschichtung kleine Zellaggregate von MD-iPSCs in iPSC Medium (ergänzt mit 5 uM Y27632, Rho-Kinase-Inhibitor ) auf frischen Matrigel (10 ug / cm 2) beschichtete 12-Well-Platte. Ändern iPSC Medium täglich. </li><li> Voll Umprogrammierung jedes Klons kann sowohl durch Genexpressionsanalyse OSKM Gene und immunhistochemische Färbung von Pluripotenzmarker (Oct3 / 4 und TRA-1-81) bestimmt werden. Eine rigorose Bewertung der Pluripotenz Potenzial sollte eingesetzt werden, um zu bestätigen vollständig umprogrammiert iPSC Linien lassen sich in drei Keimblätter differenzieren. Dies kann entweder durch Embryoidkörper (EB) die Bildung und Differenzierung Assay oder einem ter erreicht werdenatoma Bildungstest. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Modellierung von Herzkreislauferkrankungen mit iPS-Zellen entwickelt sich zu einem gemeinsamen Ansatz für den genetischen Beitrag 4-6 zu verstehen. Einige unerwartete Schwierigkeiten, die einem Zellproben von Patienten, insbesondere junge Kinder, können, indem sie die Möglichkeit einer nichtinvasiven Ansatz wie einer Urinsammlung vermieden werden. In diesem jungen Patientengruppe ist es oft schwierig, eine Urinvolumen ausreichend zu sammeln, um genügend Zellen zur Reprogrammierung Urin ergeben. Coaching d...

Kardiomyopathie ist die zweithäufigste Todesursache bei Patienten mit Duchenne und Becker-Muskeldystrophie (MD). Obwohl Mutationen in dem X-verknüpften Dystrophin-Gen in 1 auftreten: 3,500 männlichen Neugeborenen, ist sehr wenig über die molekularen und zellulären Ereignisse, die zum fortschreitenden Herzmuskelschäden bekannt. Menschen induzierte pluripotente Stammzellen von Muskeldystrophie-Patienten stammen, wurden als neuartige Instrument, um die zugrunde liegenden Krankheitsmechanismen zu studieren und für Wirkstoff-Screening 1,2 verwenden entstanden. Die erwartete Beschwerden von Hautbiopsien oder Blutproben kann jungen Patienten und / oder ihrer Erziehungsberechtigten davon abzubringen, die Einwilligung zur Studienteilnahme zu geben. Urinproben ein nicht-invasives Quelle somatischer Zellen, abänderbar Neuprogrammierung Methoden. Wir haben kürzlich gezeigt, dass Urin Zellen von Muskeldystrophie Patienten gesammelt wurden, können kultiviert werden und effizient in iPSCs Hilfe retroviraler Transduktion mit den Yamanaka Faktoren (umprogrammiertOCT3 / 4, Sox2, Klf4 und c-Myc; OSKM) 1. Der Nachteil der retroviralen Gentransfer ist die zufällige Integration der Umprogrammierung Gene in die Wirtschromosomen. Um diese Einschränkung zu überwinden, haben wir das nicht-integrierenden Sendaivirus zur Urin Reprogrammierung verwendet. Dieses Protokoll beschreibt die Sendai-Virus Reprogrammierung von Zellen aus Urin isoliert Muskeldystrophie-Patienten, die dann in Herzmuskelzellen oder anderen Zelltypen für eine weitere Studie unterschieden werden können. Dieses Protokoll kann auch für andere Patienten spezifischen Krankheiten angepasst werden.

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1144px;">List of Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 Oz. Specimen Cup with Lid</td>
			<td>STL Medical Supply</td>
			<td>M9AMSAS340</td>
			<td>For Urine Sample Collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round Glass Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-81</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CytoTune-iPS Sendai Reprogramming Kit</td>
			<td>Life Technologies</td>
			<td>A1378-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen-Strep w/o Glutamine&#160;</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/o Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>0050129SA</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 (1:1)</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene, 1:5000&#160;</td>
			<td>Life Technologies</td>
			<td>15040066</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bFGF (10 &#181;g)</td>
			<td>Life Technologies</td>
			<td>13256-029</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counter Cartridges</td>
			<td>Life Technologies</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum (500 mL Bottle)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recovery Cell Culture Freezing Medium</td>
			<td>Life Technologies</td>
			<td>12648-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, High Glucose, Glutamax</td>
			<td>Life Technologies</td>
			<td>10566-016</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ham&#39;s F-12 Nutrient Mix&#160;</td>
			<td>Life Technologies</td>
			<td>11765-054</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGF Recombinant Human Protein, Liquid Form</td>
			<td>Life Technologies</td>
			<td>PHG0311L</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin, Human Recombinant, Zinc Soln</td>
			<td>Life Technologies</td>
			<td>12585-014</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TeSR-E8</td>
			<td>Stem Cell Technologies</td>
			<td>5940</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ROCK Inhibitor (Y-27632)</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>hESC Qualified Matrix, LVED Free</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>170-8890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DreamTaq Green PCR Master Mix (2X)</td>
			<td>Thermo-Scientific</td>
			<td>K1081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS-Qualified</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ADENINE BIOREAGENT</td>
			<td>Sigma Aldrich</td>
			<td>A2786-5G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHOLERA TOXIN FROM VIBRIO CHOLERAE</td>
			<td>Sigma Aldrich</td>
			<td>C8052-.5MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HYDROCORTISONE</td>
			<td>Sigma Aldrich</td>
			<td>H0888-1G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HOLO-TRANSFERRIN FROM HUMAN</td>
			<td>Sigma Aldrich</td>
			<td>T0665-50MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,3&#39;,5-TRIIODO-L-THYRONINE SODIUM SALT&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T6397-100MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-3598</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluoromount Aquous mounting medium</td>
			<td>Sigma Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% Paraformaldehyde</td>
			<td>Alfa-Aesar</td>
			<td>43368</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">List of Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD44 - labelled with FITC&#160;</td>
			<td>BD Biosciences</td>
			<td>560977</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD146 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD73 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-&#945;-smooth muscle actin</td>
			<td>Sigma Aldrich</td>
			<td>A2547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-Vimentin</td>
			<td>Abcam</td>
			<td>ab8978-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Anti - TRA-1-81</td>
			<td>Life Technologies</td>
			<td>411100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti Oct 3/4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-9081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:415px;">Mouse Anti Dystrophin</td>
			<td>Leica Biosystems</td>
			<td>NCL-DYSB</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells

Dystrophischen Kardiomyopathie ist eine schlecht verstanden Folge der Muskeldystrophie. Generieren von induzierten pluripotenten Stammzellen (iPS-Zellen) von Patienten mit Muskeldystrophie ist eine unschätzbare Quelle für zelluläre in vitro Krankheitsmodellsysteme und für Wirkstoff-Screening-Studien verwendet werden. Patienten stammenden Urinzellen in erfolgreiche Reprogrammierung in induzierte pluripotente Stammzellen, um dystrophischen Kardiomyopathie Muster 1 verwendet. Adressieren der Sicherheitsbedenken von integrierenden Vektor-Systeme, unter Verwendung eines nicht-integrierenden Sendai-Virus-Vektors für die Transduktion von Yamanaka Faktoren in den Urin-Zellen von Patienten mit Muskeldystrophie gesammelt präsentieren wir ein Protokoll. Dieses Protokoll erzeugt vollständig neu programmiert Klone innerhalb von 2-3 Wochen. Die pluripotenten Zellen sind vektorfreien Durchgang durch-13. Diese dystrophischen iPS-Zellen lassen sich in Herzmuskelzellen differenziert und entweder verwendet werden, um Krankheitsmechanismen und für Wirkstoff-Screening zu untersuchen.

Meist Progenitorzellen aus menschlichem Urin isoliert sind positiv für uroepithelial Vorläufer und Perizyten Marker wie CD44, CD73 und CD146 (97,37%, 97,09% und 97,3% jeweils, Figur 1A und 1B). Diese Zellen exprimierten auch andere mesenchymalen Marker wie alpha-Aktin der glatten Muskulatur und Vimentin (1B). RT-PCR-Analyse zeugt von einer gemischten Population von Zellen in den Kulturen, daß es eine schwache Expression von Cytokeratin-7 (CK-7) und Uroplakin (UP) -Ia...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells at JoVE.com

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Erzeugung von induzierten pluripotenten Stammzellen von Muskeldystrophie-Patienten: Effiziente Integration freie Neuprogrammierung der Urin-abgeleiteten Zellen

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

generation-induced-pluripotent-stem-cells-from-muscular-dystrophy

Research

JoVE Journal

Medicine

11.1K Aufrufe.  Medical College of Wisconsin. This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction. 

Serie: Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolation, Charakterisierung und vergleichende Differenzierung humaner Dental Pulp Stammzellen aus bleibenden Zähne mit zwei verschiedenen Methoden abgeleitet

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Forschung

Medizin

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Patienten abgeleitet Zellkultur und Isolierung von CD133<sup&gt; +</sup&gt; Mutmaßliche Cancer Stem Cells von Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studieren Bauchspeicheldrüsenkrebs Stammzelleigenschaften für die Entwicklung neuer Behandlungsstrategien

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generation von Prostatakrebs-Patienten gewonnen Xenograft-Modelle von zirkulierenden Tumorzellen

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

Modellierung Muskeldystrophie 1 in C2C12 Myoblastenzellen

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Eine schnelle und effiziente Erzeugung von rekombinanten humanen pluripotenten Stammzellen durch Rekombinase-vermittelten Kassettenaustausch in der<em&gt; AAVS1</em&gt; Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin-induzierte Quellung in Intestinale Organoide: Ein<em&gt; In Vitro</em&gt; Assay für Fibrosis Patienten in Cystic Drug Response-Beurteilung

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

Bewertung der Exon-Aufnahme durch induzierte Splice Switching Antisense Oligonukleotide in SMA Patienten Fibroblasten

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Ex-Vivo Expansion von hämatopoetischen Stammzellen aus menschlichen Nabelschnur Blut abgeleitet CD34+ Zellen mit Valproinsäure

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...