The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. P&#233;bay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay); Australian Research Council Future Fellowship (A. P&#233;bay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.

La raccolta del campione di capelli umani da individui richiede l&#39;approvazione etica dal comitato etico della ricerca umana nelle istituzioni ospitanti e deve essere fatto in conformità con le linee guida istituzionali. 1. Isolamento dei cheratinociti da capelli a pizzico <ol><li> Disgelo matrice extracellulare soluzione (ECM) (cioè, Matrigel) su ghiaccio O / N. </li><li> Utilizzando puntali delle pipette pre-refrigerati, aggiungere 200 ml soluzione ECM per 12 ml di acqua refrigerata a medio DMEM / F12. Coat un 12-pozzetti con 1 ml di soluzione di ECM diluita in ciascun pozzetto. Incubare la piastra O / N rivestito a 37 ° C. Lavare la piastra ricoperta di pre-riscaldato DMEM / F12 medio prima dell&#39;uso. </li><li> Raccogliere i capelli umani primaria dalla testa del soggetto posizionando le dita vicino alla radice dei capelli e spennare i capelli in un movimento veloce e regolare. Controllare per confermare che ogni capelli raccolti contiene un follicolo pilifero intatta e determinare la fase di crescita delle Nazioni Unite capellider un microscopio da dissezione (Figura 1). <ol><li> Raccogliere 5-10 peli anagen da ogni individuo per estrarre cheratinociti. Processo pizzicato peli più presto possibile garantire la corretta derivazione dei cheratinociti. Nota: La Figura 1 illustra la morfologia di capelli in diverse fasi del ciclo di crescita. Capelli anagen è in fase di crescita, con uno strato esterno visibile dell&#39;epitelio circonda la lunghezza dei capelli, noto come ORS. D&#39;altra parte, i capelli telogen rappresenta capelli nella fase di riposo e non ha un ORS visibile, ma ha una chiara palla di cellule che ricoprono la radice dei capelli, noto come il rigonfiamento telogen. </li></ol></li><li> Porre i campioni di capelli in una capsula petri contenente 10 ml di antibiotico Mix per lavare per 5 min a temperatura ambiente. </li><li> Utilizzando forbici e pinze sterilizzate, tagliare eccessiva fusto del capello, lasciando un frammento di capelli con un follicolo pilifero intatto e dintorni 0,5-1 cm di fusto del capello. </li><li> Attentamente TRAnsfer frammento 1 capelli in ciascun pozzetto della piastra ECM rivestite con pinze. </li><li> Con una pipetta 1 ml, aggiungere circa 3 gocce di Knockout siero sostituzione (KSR) medio (~ 100-200 mL) ai campioni di capelli. Nota: Mantenere i frammenti di capelli a stretto contatto con la superficie della piastra per consentire l&#39;attaccamento e la successiva escrescenza cheratinociti. L&#39;aggiunta eccessiva della carta in questa fase può provoca i frammenti di capelli di galleggiare e diminuisce il rischio di attacco di capelli di successo. </li><li> Incubare i campioni di capelli in un incubatore a 37 ° con il 5% di CO 2 e consentire i follicoli dei capelli per fissare O / N. Il giorno dopo, aggiungere delicatamente altri 3 gocce di KSR media (~ 100-200 ml) per mantenere i campioni di capelli umidi. </li><li> Osservare i campioni di capelli ogni giorno per confermare che i follicoli dei capelli hanno attaccato con successo. Generalmente attaccamento dei follicoli dei capelli è evidente entro 1-3 giorni. Capelli spessi sono di solito più facili da montare rispetto a peli fini, come peli sottili hanno una maggioretendenza a galleggiare che ostacolano il processo di attaccamento. <ol><li> Una volta che i capelli sono attaccato alla piastra rivestita, aggiungere 500 ml di KSR mezzo al pozzo. Prestare particolare attenzione quando si aggiunge il mezzo come campioni di capelli potrebbero non essere montato saldamente. Da questo punto, cambiare supporto ogni 2 giorni. Nota: escrescenze cheratinociti devono essere visibili dalla regione ORS del frammento capelli dopo 3-7 giorni (Figura 2A-B). Lasciare i cheratinociti a crescere fino a 14 giorni prima passaging le cellule come descritto nella sezione successiva. </li></ol></li></ol> 2. Manutenzione e Passaging dei cheratinociti <ol><li> Pre-coat un 6-pozzetti utilizzando il kit Coating Matrix. Aggiungere 680 pl di mezzo di diluizione dal kit a ciascun pozzetto, seguito da 6,8 ml di soluzione di rivestimento Matrix. Agitare la piastra per assicurare una distribuzione uniforme della soluzione di rivestimento Matrix sulla piastra. </li><li> Il lamierino rivestito per incubare per 30 minuti a RT. Rimuoverela soluzione di rivestimento Matrix prima placcatura dei cheratinociti. </li><li> Dissociarsi cheratinociti in una sospensione di cellule singole con 500 ml di 0,25% tripsina per pozzetto per 2-5 minuti a 37 ° C. Disattivare la tripsina con 500 ml di mezzo contenente siero (ad esempio, siero fetale bovino (FBS) medium) e raccogliere in una provetta sterile da 15 ml. Rimuovere il frammento capelli con pinze sterilizzate. </li><li> Determinare il numero di cellule raccolte utilizzando un emocitometro. </li><li> Centrifugare le cellule per 3 min a 200 x g. Lavare le cellule con PBS e centrifugazione ripetere. Aspirare il PBS e risospendere il pellet cellulare in fresco medio dei cheratinociti. </li><li> Piatto giù 4 × 10 4 cheratinociti in un pozzetto della piastra ECM rivestite in presenza di cheratinociti media. Mantenere cheratinociti in un incubatore a 37 ° con il 5% di CO 2 (Figura 2C). Modificare i media ogni giorno e di passaggio celle quando la cultura è ~ 80% confluenti, WHIch può richiedere circa 2-7 giorni a seconda del numero di passaggio e la proliferazione tasso dei cheratinociti. I cheratinociti derivati ​​dovrebbero essere estesi per generare scorte congelate sufficienti utilizzando metodi di crioconservazione lento congelamento standard di 16. Nota: Le condizioni di coltura descritte supportano l&#39;espansione della popolazione omogenea dei cheratinociti con la tipica morfologia ciottoli (Figura 2C). Di tanto in tanto, qualche cheratinociti differenziati, con grande e piatta morfologia e hanno perso potenziale replicativo, possono essere osservati in cultura. Caratterizzazione dei cheratinociti può essere eseguita mediante immunocolorazione utilizzando anticorpi contro citocheratina 14 come descritto in precedenza 17. Linee cellulari di cheratinociti Fondata dovrebbero essere testati per micoplasma dopo 1-3 passaggi utilizzando il kit disponibile in commercio. </li></ol> 3. Generazione di hiPSCs da cheratinociti Utilizzando episomiale Vettori <ol><li> Pre-coat un 6-pozzetti con0,1% di gelatina (2 ml / pozzetto). Consentire la gelatina per rivestire per almeno 20 minuti a temperatura ambiente. </li><li> Per esperimento riprogrammazione, uso cheratinociti non oltre il passaggio 6. Dissociate cheratinociti in una sospensione di cellule singole con 1 ml di 0,25% tripsina per bene per 2-5 minuti a 37 ° C. Inattivare tripsina con 1 ml di supporti contenenti siero (ad esempio, FBS media) e raccogliere in una provetta da 15 ml. </li><li> Celle di centrifugazione per 3 min a 200 xg e risospendere le cellule in cheratinociti media. Piastra 1 × 10 5 -1.5 × 10 5 cheratinociti per pozzetto in una piastra O / N gelatinizzato. </li><li> Il giorno successivo (giorno 0), controllare la piastra per garantire cellule sono il 50-60% confluenti. Nota: Se cheratinociti sono meno di ~ 50% confluenti potrebbero non crescono bene dopo la trasfezione. </li><li> Il giorno 0, effettuare trasfezione dei vettori episomiale riprogrammazione sui cheratinociti, utilizzando un rapporto di 8 microlitri trasfezione reagente 2 ug di DNA plasmidico totale.<ol><li> Prendete 0,5 mg per ciascuno dei 4 plasmidi (pCXLE-eGFP, pCXLE-hOct3 / 4-shP53F, pCXLE-HSK, pCXLE-Hul) e diluire in 100 ml di media Serum ridotta (per esempio, OPTI-MEM). Nota: Il plasmide pCXLE-eGFP è utilizzato per monitorare l&#39;efficienza di trasfezione di cheratinociti e non è necessaria per la riprogrammazione successo. </li><li> Aggiungere 8 ml di reagente di trasfezione di DNA diluito. Mescolare muovendo il tubo. Lasciare la miscela di reazione DNA riposare indisturbati a temperatura ambiente per 15 min. </li><li> Sostituire il supporto dei cheratinociti con terreno fresco. </li><li> Aggiungere delicatamente la miscela di reazione del DNA di coltura di cheratinociti e incubare a 37 ° C incubatore con 5% di CO 2 per 4 ore. Nota: non è consigliabile eseguire prolungata trasfezione O / N nei cheratinociti in quanto presentano bassa sopravvivenza dopo la trasfezione. </li><li> Dopo 4 ore dopo la trasfezione, sostituire il mezzo di cheratinociti con terreno fresco. </li></ol></li><li> Il giorno 1 (1 giorno post-transfection), cambiare il mezzo cheratinociti e verificare l&#39;efficienza di trasfezione stimando la percentuale di cellule positive eGFP sotto un microscopio a fluorescenza. Tipicamente&gt; 60-70% efficienza di trasfezione si osserva per cheratinociti primari che utilizzano questo protocollo. </li><li> Il giorno 2, ripetere trasfezione come descritto al punto 3.5. Due trasfezioni consecutivi sarebbero tipicamente raggiungere il&gt; 90% efficienza di trasfezione. Coltura di cheratinociti solito raggiunge la confluenza prima della fine del secondo trasfezione. <ol><li> Preparare un piatto 10 centimetri di topo alimentatore fibroblasti embrionali (MEF) per ogni reazione riprogrammazione. Preparare alimentatori MEF mitoticamente inattivati ​​come descritto in precedenza 18. </li><li> Pre-coat un piatto 10 centimetri con 5 ml di 0,1% di gelatina per almeno 20 min. Tavola 4 × 10 6 MEF mitoticamente inattivato per 10 cm piatto in FBS medio. Permettono alle cellule di stabilirsi e di allegare O / N in un incubatore a 37 ° con il 5% di CO 2. </li></ol></li><li> Il giorno 3, harconferire cheratinociti con 0,25% tripsina come descritto al punto 3.2 e 3.3. Risospendere cheratinociti in KSR medio e piastra 90% dei cheratinociti da 1 bene di un 6-pozzetti in un piatto alimentatore MEF 10 centimetri con KSR media. </li><li> Dal 4 ° giorno in poi, cambia il mezzo KSR ogni giorno. <ol><li> In alternativa, dopo il giorno 18, sostituire terreno di coltura ogni due giorni. Nota: cheratinociti non-riprogrammato cesseranno di proliferare, mentre le colonie hiPSC con contorno definito che assomigliano cellule staminali embrionali umane (hESC) emergeranno dal giorno 14-21. </li></ol></li><li> Isolare colonie hiPSC da dissezione manuale dal giorno 21 in poi. Evitare colonie hiPSC che sono strettamente raggruppati insieme e solo raccolgono le colonie hiPSC che sono nettamente separati dagli altri. Nota: le colonie hiPSC possono essere presi in precedenza, ma la colonia con confini definiti potrebbero non essere evidente in precedenti momenti viste le ridotte dimensioni della colonia. Efficienze Riprogrammazione possono variare tra diffcheratinociti di erenti paziente, ed è influenzato anche da altri fattori, quali il numero di passaggio o il tasso di proliferazione dei cheratinociti. <ol><li> Per la dissezione manuale di colonie hiPSC, utilizzare un ago 26G per tagliare colonie hiPSC sotto un microscopio da dissezione. Tagliare una colonia hiPSC in piccoli pezzi intorno a 300-600 micron di lunghezza. Trasferire i pezzi da una colonia hiPSC in una nuova piastra alimentatore MEF (2,5 × 10 4 MEF / cm 2) per stabilire una linea clonale di hiPSCs. Inizialmente, la cultura ogni linea clonale hiPSC in un pozzetto di una piastra a 12 o coltura di organi e piatti, quindi espandere a 6 formato pozzetti. </li><li> Mantenere linee clonali stabilite di hiPSCs in KSR medio su alimentatori MEF. Una volta che la cultura raggiungere il 70 - 80% di confluenza con le colonie hiPSC ~ 1,5 millimetri di diametro, hiPSCs passaggio utilizzando metodi standard enzimatici passaging (Accutase, dispasi o Collagenase IV) secondo le istruzioni del produttore. </li></ol></li><li> Caratterizzare stabilito linea hiPSCs per confermare la pluripotenza e la loro capacità di differenziarsi in cellule rappresentante dei tre foglietti embrionali in vitro e / o in vivo 13,19. Inoltre, le linee hiPSC routine test stabilite per micoplasma utilizzando kit disponibili in commercio per garantire che siano privi di agenti patogeni. Ordinariamente monitorare la stabilità del genoma di linee hiPSC da cariotipo o numero di copie variazione basata su array (CNV) analisi. </li></ol>

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The authors have no conflict of interest.

Generazione di hiPSCs paziente-specifici offre un approccio unico per lo studio patogenesi nei tipi di cellule malate in vitro, e fornisce anche una piattaforma per lo screening di stupefacenti per identificare nuove molecole in grado di salvare i fenotipi di malattia. Questo approccio di modellazione malattia utilizzando hiPSCs ha dato risultati promettenti per una varietà di malattie, tra cui la sindrome del QT lungo, malattia di Huntington, il morbo di Parkinson e la sclerosi laterale amiotrofica 22.</...

La scoperta di umani cellule staminali pluripotenti indotte (hiPSCs) ha rivoluzionato il campo della medicina rigenerativa, che fornisce un metodo fattibile per la generazione di cellule staminali paziente-specifici 1-3. hiPSCs sono stati generati con successo da vari tipi di cellule somatiche, inclusi i fibroblasti, cellule ematopoietiche 4,5 6,7, cellule epiteliali renali di urina 8 e cheratinociti 9,10. Fino ad oggi, i fibroblasti della pelle e cellule ematopoietiche rappresentano le fonti di cellule più comunemente usati per la generazione iPSCs paziente-specifici. Probabilmente, questo è dovuto al fatto che le biopsie cutanee e raccolta del sangue sono procedure mediche di routine e grandi biobanche di sangue o di pelle campioni di pazienti sono stati stabiliti in molti paesi. A differenza di cellule del sangue e fibroblasti cutanei che richiedono metodi di estrazione invasivi, cheratinociti rappresentano un tipo di cellula facilmente accessibile per la generazione hiPSC. Keratinocytes sono cellule epiteliali ricchi di cheratina che formano la barriera epidermica esterno della pelle e si trovano anche in unghie e capelli 11. In particolare, cheratinociti possono essere trovati sulla guaina esterna della radice (ORS) dei follicoli piliferi, uno strato cellulare esterno che copriva il fusto del capello insieme con la guaina interna della radice (IRS) celle (12, figura 1). Come la raccolta dei capelli è una procedura semplice che non richiede l&#39;assistenza di personale medico, si prevede la possibilità per i pazienti di recuperare ed inviare i propri campioni di capelli ai laboratori, il che faciliterà enormemente la raccolta dei campioni dei pazienti per la generazione hiPSC. Cheratinociti epidermici hanno una maggiore efficienza riprogrammazione e cinetica riprogrammazione più veloci rispetto ai fibroblasti, aggiungendo i vantaggi di utilizzare cheratinociti come le cellule di partenza per hiPSC generazione 9,13. Inoltre, hiPSCs può essere generato anche utilizzando altre popolazioni di cellule all&#39;interno del follicolo pilifero,comprese le cellule della papilla dermica situate alla base del 14,15 follicolo pilifero. Precedenti rapporti di generazione IPSC utilizzando cellule dei capelli derivate utilizzano spesso metodi di riprogrammazione retrovirali o basati lentivirali-9,14,15. Tuttavia, questi metodi virali introducono integrazione genomica indesiderabile dei transgeni esteri durante la riprogrammazione. In confronto, l&#39;uso di vettori episomiale rappresenta un metodo di riprogrammazione fattibile, non virale per generare iPSCs senza integrazione 4. Abbiamo già messo a punto un metodo semplice, economico e non virale per riprogrammare efficientemente cheratinociti in hiPSCs utilizzando vettori episomiale 13. Qui forniamo un protocollo dettagliato per la generazione di hiPSCs cheratinociti derivate, compresa la derivazione di cheratinociti da capelli strappato, espansione e manutenzione dei cheratinociti e successiva riprogrammazione per generare hiPSCs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic Mix:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 ng/ml Antimycotic amphotericin B</td>
			<td>Sigma</td>
			<td>A2942-20ml</td>
			<td>Antibiotic mix is made up in PBS.&#160;</td>
		</tr>
		<tr>
			<td>1X Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (-)</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR) medium:&#160;</td>
			<td></td>
			<td></td>
			<td>KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use.</td>
		</tr>
		<tr>
			<td>20% knockout serum replacement (KSR)</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with glutamax</td>
			<td>Invitrogen</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; MEM non-essential amino acid</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.5&#215; Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.1 mM &#946;-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;bFGF (10 ng/ml, added fresh)</td>
			<td>Millipore</td>
			<td>GF003</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte medium:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;EpiLife with 60&#160;&#181;M Calcium</td>
			<td>Invitrogen</td>
			<td>M-EPI-500-CA</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; Human keratinocyte growth supplement (HKGS)</td>
			<td>Invitrogen</td>
			<td>S-001-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS) medium:&#160;</td>
			<td></td>
			<td></td>
			<td>FBS medium is filtered using Stericup (Millipore, #SCGPU05RE)&#160; before use.</td>
		</tr>
		<tr>
			<td>10% fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Invitrogen</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5&#215; Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mM L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular Matrix (ECM):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning&#160;</td>
			<td>354234</td>
			<td>Aliquot Matrigel stock and store in -80&#176;C following manufacturer&#8217;s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200&#181;l matrigel for coating a 12-well plate (~150&#181;g/well).&#160;</td>
		</tr>
		<tr>
			<td>Coating Matrix Kit&#160;</td>
			<td>Invitrogen</td>
			<td>R-011-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids:&#160;</td>
			<td></td>
			<td></td>
			<td rowspan="5">Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-eGFP</td>
			<td>Addgene</td>
			<td>27082</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hOct3/4-shP53F</td>
			<td>Addgene</td>
			<td>27077</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hSK</td>
			<td>Addgene</td>
			<td>27078</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hUL</td>
			<td>Addgene</td>
			<td>27080</td>
		</tr>
		<tr>
			<td>Transfection reagent Fugene HD</td>
			<td>Promega</td>
			<td>E231B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin (from porcine skin)</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td>Make up 0.1% gelatin in distilled water. Autoclave before use.&#160;</td>
		</tr>
		<tr>
			<td>Reduced Serum medium: OPTI-MEM</td>
			<td>Invitrogen</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblast (MEF) feeder</td>
			<td></td>
			<td></td>
			<td>MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16.</td>
		</tr>
		<tr>
			<td>26G needle</td>
			<td>Terumo</td>
			<td>NN2613R</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate (tissue culture treated)</td>
			<td>BD Biosciences</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate (tissue culture treated)</td>
			<td>BD Biosciences</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dish (tissue culture treated)</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Invitrogen</td>
			<td>17105-041</td>
			<td>Use at 10mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
			<td>Use at 1mg/ml</td>
		</tr>
		<tr>
			<td>TRA-160 antibody</td>
			<td>Millipore</td>
			<td>MAB4360</td>
			<td>Use at 5&#181;g/ml</td>
		</tr>
		<tr>
			<td>OCT4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>Use at 5&#181;g/ml</td>
		</tr>
		<tr>
			<td>NANOG antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1997</td>
			<td>Use at 10&#181;g/ml</td>
		</tr>
		<tr>
			<td>MycoAlert Detection kit</td>
			<td>Lonza</td>
			<td>LT07-418</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes

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

I capelli passa attraverso 3 diverse fasi del ciclo di sviluppo: anagen (fase di crescita), catagen (la fase di regressione) e telogen (fase di riposo) il 20,21. Il follicolo pilifero anagen contiene più strati di epitelio; questi strati comprendono i ORS, IRS e il fusto del capello (Figura 1). Capelli Anagen alla fine subisce la transizione alla fase catagen, che è caratterizzato da apoptosi delle ORS e alla conclusione della differenziazione fusto del capello. Infine, la transizione capel...

Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

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

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

Generazione di cellule staminali Integrazione-libero umano pluripotenti indotte Utilizzando capelli derivati ​​da cheratinociti

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

generation-integration-free-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

11.4K Views.  University of Melbourne. This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors. 

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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Generazione di pluripotenti indotte cellule staminali da congelata Buffy cappotti con non-integrazione episomiale Plasmidi

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Ricerca

Biologia dello sviluppo

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Generazione di pluripotenti indotte cellule staminali da umani periferici T Cellule con Sendai virus in condizioni di libera-Feeder

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Generazione di cellule staminali pluripotenti indotte-Uso sinoviociti fibroblasti-come isolato da Giunti dell&#39;artrite reumatoide pazienti

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Generazione di cellule staminali pluripotenti indotte da melanoma umano Linfociti infiltranti il ​​tumore

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Pluripotenti indotte generazione di cellule staminali da cellule del sangue Utilizzando Sendai Virus e centrifugazione

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generazione di integrazione-liberi cellule staminali pluripotenti indotte dalle cellule mononucleari di sangue periferico umano Utilizzando episomiale Vettori

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Definito e Generazione scalabile di cellule epatociti-come da cellule staminali umane pluripotenti

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Derivazione libera dell&#39;integrazione delle cellule staminali pluripotenti indotte dall&#39;uomo usando la matrice Laminina 521

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Generazione di standardizzato e riproducibile del Forebrain-tipo Organoids cerebrale da umani cellule staminali pluripotenti indotte

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

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

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

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

Utilizzando umani indotti dell'epatocita-come di derivati da cellule staminali pluripotenti per scoperta della droga

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

Generazione di cellule staminali Integrazione-libero umano pluripotenti indotte Utilizzando capelli derivati ​​da cheratinociti

Generazione di cellule staminali Integrazione-libero umano pluripotenti indotte Utilizzando capelli derivati da cheratinociti