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.

NOTA: I pazienti e / oi loro tutori dovrebbero dare il consenso informato a partecipare a un Institutional Review Board ha approvato lo studio. 1. Tamponi e preparati media <ol><li> Tampone di lavaggio: Per preparare 100 ml di soluzione di lavaggio, aggiungere 1 ml di 100x penna / Soluzione strep (100 U / ml penicillina + 100 mg / ml di streptomicina) a 99 ml di soluzione fisiologica tampone fosfato (PBS). </li><li> Urinario cellule progenitrici (UPC) Media: Per preparare UPC media, mescolare volumi uguali di cheratinociti siero libero (KSF) Medium + Progenitor cellulare Medium. <ol><li> KSF Medium: Per preparare media dei cheratinociti, aggiungere 5 ng / ml di fattore di crescita epidermico (EGF), 50 ng / ml di estratto pituitario bovino (BPE), 30 ng / ml di tossina del colera, e la soluzione penna / strep (100 U / ml di penicillina, 100 mg / ml di streptomicina) a 500 ml medie KSF. </li><li> Progenitor cellulare Medium: Aggiungere tre quarti di DMEM con un quarto dei mezzi Ham-F12 e integrare con il 10% FBS, 0,4 mg / ml idrocortisone, 0,1nM tossina del colera, 5 ng / ml di insulina, 1,8 x 10 -4 M adenina, 5 mg / ml di transferrina, 2 x 10 -9 M triiodo thyronine, 10 ng / ml EGF, e la penna / strep soluzione. </li></ol></li><li> hES Media: Aggiungi il 20% di sostituzione KO siero (K-SR), aminoacidi non essenziali 1x MEM, 2 mm L-glutammina, 100 micron β-mercaptoetanolo, 20 ng / ml bFGF e penna / strep a 400 ml DMEM / F12. </li></ol> 2. Campione di urine Collection <ol><li> Istruire i pazienti di bere liquidi 30 min prima della raccolta delle urine per garantire che una quantità adeguata (~ 30-40 ml) di urina possono essere raccolti. </li><li> Dare i pazienti e / o dei loro tutori un kit di raccolta delle urine, contenente i seguenti elementi: (1) istruzioni scritte su come ottenere campione di urina cattura sterile o pulite, (2) toilettes antibatteriche umide, e (3) un 100 ml sterile bicchiere di raccolta dei campioni. Informare i pazienti di raccogliere campioni. </li><li> Porre immediatamente i campioni di urina sul ghiaccio e trasferimento laboraTory in un dispositivo di raffreddamento. Processo immediatamente l&#39;urina per l&#39;isolamento delle cellule. Se il ritardo è inevitabile, conservare i campioni in ghiaccio per un massimo di 4 ore con una minima perdita di vitalità cellulare. </li></ol> 3. Isolamento ed espansione di cellule urina NOTA: eseguire le seguenti operazioni in condizioni di sterilità in un BSL2 Cappa di sicurezza biologica. <ol><li> Usando pipette sterili, i campioni di urina trasferimento sterilizzato provette da 50 ml. Centrifugare a 400 xg per 10 min a RT. </li><li> Aspirare il surnatante lasciando 1 ml nel tubo; fare attenzione a non disturbare il pellet. </li><li> Risospendere e combinare i pellets da più tubi in 7 ml di tampone di lavaggio e ripetere centrifugazione a 400 xg per 10 min a RT. </li><li> Con attenzione aspirare il surnatante, lasciando il pellet e ~ 0,2-0,5 ml di surnatante. Risospendere in 2-3 ml di urinario Progenitor cellulare (UPC) di medie e trasferire la sospensione in 4-6 pozzetti di una nonrivestito 24-pozzetti. </li><li> Dopo 72 ore, integrare la cultura con 0,5 ml di fresco UPC medie per bene e cambiare media ogni 2-3 giorni dopo. Gli eritrociti e cellule squamose che non si attaccano alla piastra saranno rimossi con il mezzo modifiche. </li><li> Monitorare cultura dopo 4-6 giorni. NOTA: Piccole 2-4 colonie di cellule inizieranno ad apparire. Le cellule saranno arrotondati o allungata, che rappresentano di tipo I o di tipo II di epiteliale renale (RE) le cellule rispettivamente 3. </li><li> Cambiare la media ogni 2-3 giorni dal momento che, una volta compaiono le cellule, che sarà oggetto di una fase di rapida espansione. </li><li> Una volta che le cellule raggiungono 80-90% di confluenza, circa 9-15 giorni dopo la placcatura, dissociare e il passaggio delle cellule (15,000-18,000 cellule / cm 2) sul 2-4 pozzetti di 24 pozzetti per un&#39;ulteriore espansione. Segna questo come cellulare di passaggio 1 (P1). </li><li> Caratterizzare cellule di urina per le specifiche lignaggio attraverso l&#39;analisi di citometria di flusso, immunohistochemcolorazione ical o tramite reazione inversa a catena transcriptasi-polimerasi (RT-PCR). </li><li> Riprogrammare cellule urina isolate (sezione 4) o congelare giù a congelamento (DMEM integrato con il 10% di siero e il 10% DMSO) per crioconservazione a lungo termine. </li></ol> 4. Urine Riprogrammazione cellulare Utilizzando Sendai Virus NOTA: La corretta gestione e l&#39;uso di DPI (dispositivi di protezione individuale) è raccomandato durante la manipolazione degli agenti trasfettando. Effettuare le seguenti operazioni in condizioni sterili in un BSL2 Biological Safety Cabinet. Il corretto smaltimento dei trasfezione cellule agente e / o transfettate si consiglia di evitare il rischio di pericoli per l&#39;ambiente e per la salute. Per urine riprogrammazione cellulare, utilizzare un Sendai Riprogrammazione Kit con modifiche al protocollo alimentatore-dipendente dal produttore, come descritto di seguito. <ol><li> Per garantire un&#39;elevata efficienza riprogrammazione utilizzando il Sendai, uso passaggi 1-5 di cellule di urina che sono rapidily dividendo. Se le cellule non replicano bene o diventano senescenti, disfarsene in quanto non riprogrammare in modo efficiente. </li><li> Seed 60.000 cellule per pozzetto in due pozzetti di una piastra 6 bene. Segna questo come giorno -2. Regolare la densità di semina delle cellule (5 x 4-9 OTTOBRE x 10 4 cellule per pozzetto) come necessario per raggiungere 80-90% di confluenza di giorno 0. </li><li> Il giorno 0 (48 ore dopo la placcatura le cellule), preparare i vettori di riprogrammazione Sendai (SEV) contenenti ciascuno dei quattro fattori OSKM. Aggiungere ognuno dei vettori a 1 ml di pre-riscaldato a medio UPC. Utilizzare un MOI di 1-1,5 (5-7.5 x 10 5 CIU), che è sufficiente per cellule urina riprogrammazione. Aspirare il medio e aggiungere lentamente il 1 ml UPC + SeV ad un pozzetto delle celle di urina. Utilizzare il secondo e il controllo negativo trasduzione. </li><li> Il giorno 1, sostituire il mezzo di UPC + SeV fresca media UPC. Alcuni morte cellulare è visto dopo 24 ore a causa di citotossicità virale. </li><li> A seconda dell&#39;efficienza di clon riprogrammatoe formazioni e la morfologia compatta, continuano a cambiare il mezzo di tutti i giorni fino al giorno 6 o 7. </li><li> Un giorno prima della dissociazione di cellule trasdotte (giorno 5 o 6), preparare piastre alimentatore MEF placcando mitomicina-C (10 ug / ml per 3 ore) trattati-MEF a densità di 5 x 10 4 cellule / cm 2, su 0.1 % di gelatina rivestito 100 mm 2 piatti di coltura. </li><li> Il giorno successivo (giorno 6 o 7), dissociare le cellule con 0,25% tripsina, risospendere le cellule in UPC medio e piastra 5 x 10 4 - Piastra alimentatore 2 x 10 5 cellule / 100 mm 2 MEF. </li><li> Il giorno seguente, passare hES medie e cambiare il mezzo ogni giorno. Osservare le cellule al microscopio per monitorare le cellule trasformate. NOTA: Le cellule formano aggregati clonali con morfologia caratteristica ciottoli e avere maggiore nucleo rapporto citoplasmatica (12-18 giorni dopo trasduzione). </li><li> Entro due o tre settimane dopo la trasduzione, raccogliere le colonie e il trasferimento di nuove piastre per clonespansione al. </li><li> Eseguire colorazione delle cellule in tempo reale per la selezione dei cloni IPSC incubando le cellule con TRA-1-81 anticorpi per 1 ora a 37 ° C in incubatore CO 2 seguita dal contatore colorazione con colorante fluorescente specifico anticorpo secondario coniugato per 1 ora a 37 ° C in incubatore CO 2. </li><li> Utilizzare un pollice G 1½ ago 26 a tratteggio incrociato più grandi TRA-1-81 positivi cloni IPSC in piccoli pezzi uguali dimensioni (4-6 pezzi per clone). </li><li> Utilizzare una pipetta punta sterile per raccogliere e trasferire 10-20 pezzi cross-nati da più cloni in tutti i pozzetti di Matrigel (10 mg / cm 2) Rivestiti 24 pozzetti con HES media. NOTA: Pezzi di un singolo clone placcato singolarmente in un unico pozzo non si espandono o mantenere pluripotenza. </li><li> Cambiare dal hES all&#39;IPSC medio 24-48 ore dopo i cloni riprogrammati sono attaccati alla superficie Matrigel. </li><li> Durante la manutenzione su piastre Matrigel rivestite, SCRA manualmentepe-off tutte le cellule differenziate o contaminanti cellule di alimentazione MEF con una pipetta punta ad arricchire per distrofici iPSC (MD-IPSC) cloni pienamente riprogrammate e pluripotenti. </li><li> Dopo i singoli cloni hanno raggiunto ~ 100 micron dimensioni, espandere i cloni dissociandosi con EDTA 0,48 mm (etilendiamminotetraacetico) soluzione per 3 min e placcatura piccoli aggregati di cellule di MD-iPSCs sospese in iPSC medio (integrato con 5 micron Y27632, Rho Kinase inibitore ) su Matrigel freschi (10 mg / cm 2) rivestito 12 pozzetti. Cambiare iPSC media giornaliera. </li><li> Riprogrammazione completa di ogni clone può essere determinata sia da analisi di espressione genica di geni OSKM e colorazione immunoistochimica di marcatori pluripotenza (Oct3 / 4 e TRA-1-81). Una valutazione rigorosa del potenziale pluripotenza dovrebbe essere impiegato per confermare le linee IPSC completamente riprogrammati in grado di differenziare in tre foglietti embrionali. Ciò può essere eseguito mediante corpo embrioide (EB) formazione e differenziazione dosaggio o terAtoma test formazione. </li></ol>

<ol>
	<li>Guan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin-deficient+cardiomyocytes+derived+from+human+urine:+New+biologic+reagents+for+drug+discovery.">Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery.</a> Stem Cell Research. 12 (2), 467-480 (2014).</li><li>Dick, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon+Skipping+and+Gene+Transfer+Restore+Dystrophin+Expression+in+Human+Induced+Pluripotent+Stem+Cells-Cardiomyocytes+Harboring+DMD+Mutations.">Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations.</a> Stem Cells Dev. 22 (20), 2714-2724 (2013).</li><li>Zhou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+urine+samples.">Generation of human induced pluripotent stem cells from urine samples.</a> Nat Protoc. 7 (12), 2080-2089 (2012).</li><li>Bellin, M., Marchetto, M. 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The authors declare that they have no competing financial interests.

Modellazione malattie cardiovascolari utilizzando iPSCs sta diventando un approccio comune per capire il contributo genetico 4-6. Alcune difficoltà impreviste di ottenere campioni di cellule da pazienti, soprattutto bambini, possono essere evitati, offrendo la possibilità di un approccio non invasiva come una raccolta di urina. In questo giovane popolazione di pazienti, è spesso difficile raccogliere un volume di urina sufficiente per produrre cellule urina sufficiente per la riprogrammazione. Coaching i p...

Cardiomiopatia è la seconda causa di morte nei pazienti con Duchenne e Becker distrofia muscolare (MD). Sebbene mutazioni nel gene della distrofina X-linked si verificano in 1: 3.500 nati maschi, si sa molto poco circa gli eventi molecolari e cellulari che portano alla progressiva danni muscolo cardiaco. Cellule staminali pluripotenti indotte umane derivate da pazienti distrofia muscolare sono emersi come un nuovo strumento per studiare i meccanismi della malattia sottostante e da utilizzare per lo screening di stupefacenti 1,2. Il disagio anticipata di biopsie cutanee o campioni di sangue possono dissuadere i pazienti giovani e / oi loro tutori a dare il consenso per la partecipazione allo studio. I campioni di urina sono una fonte non invasiva di cellule somatiche che sono modificabili a metodi di riprogrammazione. Abbiamo recentemente dimostrato che le cellule di urina raccolti da pazienti distrofia muscolare possono essere colta ed efficiente riprogrammati in iPSCs utilizzando trasduzione retrovirale con i fattori di Yamanaka (Oct3 / 4, Sox2, Klf4, e c-Myc; OSKM) 1. Lo svantaggio di consegna del gene retrovirale è l&#39;integrazione casuale dei geni riprogrammazione nei cromosomi dell&#39;ospite. Per superare questo limite, abbiamo usato il Sendai non-integrazione per la riprogrammazione delle cellule urine. Dettagli Questo protocollo virus riprogrammazione Sendai di cellule di urina isolate da pazienti distrofia muscolare, che possono poi essere differenziati in cardiomiociti o altri tipi di cellule per ulteriori studi. Questo protocollo può anche essere adattato per altre malattie specifiche del paziente.

<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

Cardiomiopatia distrofico è una conseguenza poco conosciuta di distrofia muscolare. Generazione di cellule staminali pluripotenti indotte (iPSCs) di pazienti con distrofia muscolare è una fonte preziosa per cellulari in vitro sistemi modello di malattia in e può essere utilizzato per gli studi di screening di stupefacenti. Cellule urina paziente derivate sono stati utilizzati in riprogrammazione successo in cellule staminali pluripotenti indotte per modellare cardiomiopatia distrofica 1. Affrontare i problemi di sicurezza di integrazione di sistemi vettoriali, vi presentiamo un protocollo con un non-integrazione di Sendai virus vettore per la trasduzione di Yamanaka fattori in cellule di urina raccolti da pazienti affetti da distrofia muscolare. Questo protocollo genera completamente riprogrammato cloni entro 2-3 settimane. Le cellule pluripotenti sono esenti da vettore per il passaggio-13. Questi iPSCs distrofiche possono essere differenziati in cardiomiociti e utilizzati sia per studiare i meccanismi di malattia o per lo screening di stupefacenti.

La maggior parte delle cellule progenitrici isolate da urina umana sono positivi per uroepithelial progenitrici e periciti marcatori quali CD44, CD73 e CD146 (97.37%, 97.09% e 97,3% rispettivamente; la figura 1A e 1B). Queste cellule hanno espresso altri marcatori mesenchimali come alfa-actina del muscolo liscio e vimentina (Figura 1B). RT-PCR dà prova di una popolazione mista di cellule nelle culture che non è debole espressione di Cytokeratin-7 (CK-7) e Uroplakin (U...

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.

Generazione di pluripotenti indotte cellule staminali da distrofia muscolare di pazienti: Efficient Riprogrammazione senza integrazione di origine urinaria Cells

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

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Research

JoVE Journal

Medicine

11.1K Views.  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. 

Video: 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.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

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

Ricerca

Medicina

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).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di 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.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

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.

Generazione di cancro alla prostata del paziente Derivato modelli di xenotrapianto di cellule tumorali circolanti

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.

Modellazione Distrofia miotonica 1 nelle cellule dei mioblasti C2C12

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.

Generazione rapida ed efficace delle ricombinante cellule staminali umane pluripotenti da ricombinasi mediata scambio Cassette in<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 indotta Gonfiore intestinale organoidi: An<em&gt; In Vitro</em&gt; Test per la valutazione della risposta farmacologica nella fibrosi cistica pazienti

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.

Valutazione dell'inclusione dell'esone indotta da impiombare commutazione oligonucleotidi antisenso in fibroblasti del paziente di SMA

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 espansione di cellule staminali da cordone ombelicale umano derivate dal sangue CD34+ cellule utilizzando acido valproico

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).

Isolamento e cultura della miofibra singola da un modello Murine della distrofia muscolare di Emery-Dreifuss nei primi sviluppi post-Natal

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