Name: generation of induced pluripotent stem cells from frozen buffy coats using non-integrating episomal plasmids
Uploaded: 2015-06-05T14:00:00
Description: Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids at JoVE.com

The study was supported by the Ministry of Health and Department of Educational Assistance, University and Research of the Autonomous Province of Bolzano and the South Tyrolean Sparkasse Foundation.

Cellule mononucleari del sangue periferico (PBMNCs) sono stati isolati da campioni di sangue periferico di donatori sani, dopo firmato il consenso informato e l&#39;approvazione del Comitato Etico della Provincia di Bolzano. Gli esperimenti sono stati condotti in conformità con i principi espressi nella Dichiarazione di Helsinki. Tutti i dati sono stati raccolti e analizzati in modo anonimo. 1. Isolamento di cellule mononucleate del sangue periferico (PBMNCs) <ol><li> PBMNCs ottenuti da b...

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Takahashi, K., Okita, K., Nakagawa, M., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+fibroblast+cultures.">Induction of pluripotent stem cells from fibroblast cultures.</a> Nat. Protoc. 2 (12), 3081-3089 (2007).</li><li>Drews, K., Jozefczuk, J., Prigione, A., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells--from+mechanisms+to+clinical+applications.">Human induced pluripotent stem cells--from mechanisms to clinical applications.</a> J. Mol. Med. (Berl). 90 (7), 735-745 (2012).</li><li>Bayart, E., Cohen-Haguenauer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technological+overview+of+iPS+induction+from+human+adult+somatic+cells).">Technological overview of iPS induction from human adult somatic cells).</a> Curr. Gene Ther. 13 (2), 73-92 (2013).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Sommer, A. G., 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+peripheral+blood+using+the+STEMCCA+lentiviral+vector.">Generation of human induced pluripotent stem cells from peripheral blood using the STEMCCA lentiviral vector.</a> J. Vis. Exp. (68), e4327 (2012).</li><li>Zhou, W., Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+gene+delivery+can+reprogram+human+fibroblasts+to+induced+pluripotent+stem+cells.">Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.</a> Stem Cells. 27 (11), 2667-2674 (2009).</li><li>Lieu, P. T., Fontes, A., Vemuri, M. C., Macarthur, C. C. <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+induced+pluripotent+stem+cells+with+CytoTune,+a+non-integrating+Sendai+virus.">Generation of induced pluripotent stem cells with CytoTune, a non-integrating Sendai virus.</a> Methods Mol. Biol. 997, 45-56 (2013).</li><li>Woltjen, K., 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=piggyBac+transposition+reprograms+fibroblasts+to+induced+pluripotent+stem+cells.">piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.</a> Nature. 458 (7239), 766-770 (2009).</li><li>Okita, K., 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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nat. Methods. 8 (5), 409-412 (2011).</li><li>Kim, D., 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+by+direct+delivery+of+reprogramming+proteins.">Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.</a> Cell. Stem Cell. 4 (6), 472-476 (2009).</li><li>Warren, L., Ni, Y., Wang, J., Guo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+derivation+of+human+induced+pluripotent+stem+cells+with+messenger+RNA.">Feeder-free derivation of human induced pluripotent stem cells with messenger RNA.</a> Sci. Rep. 2, 657 (2012).</li><li>Stadtfeld, M., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotency:+history,+mechanisms,+and+applications.">Induced pluripotency: history, mechanisms, and applications.</a> Genes Dev. 24 (20), 2239-2263 (2010).</li><li>Chou, B. K., 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=Efficient+human+iPS+cell+derivation+by+a+non-integrating+plasmid+from+blood+cells+with+unique+epigenetic+and+gene+expression+signatures.">Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.</a> Cell Res. 21 (3), 518-529 (2011).</li><li>Kim, C., 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=Studying+arrhythmogenic+right+ventricular+dysplasia+with+patient-specific+iPSCs.">Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.</a> Nature. 494 (7435), 105-110 (2013).</li><li>Aasen, 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=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat. Biotechnol. 26 (11), 1276-1284 (2008).</li><li>Streckfuss-Bomeke, K., 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=Comparative+study+of+human-induced+pluripotent+stem+cells+derived+from+bone+marrow+cells,+hair+keratinocytes,+and+skin+fibroblasts.">Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.</a> Eur. Heart J. 34 (33), 2618-2629 (2013).</li><li>Miyoshi, N., 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=Reprogramming+of+mouse+and+human+cells+to+pluripotency+using+mature+microRNAs.">Reprogramming of mouse and human cells to pluripotency using mature microRNAs.</a> Cell. Stem Cell. 8 (6), 633-638 (2011).</li><li>Wang, Y., 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=Induced+pluripotent+stem+cells+from+human+hair+follicle+mesenchymal+stem+cells.">Induced pluripotent stem cells from human hair follicle mesenchymal stem cells.</a> Stem Cell. Rev. 9 (4), 451-460 (2013).</li><li>Beltrao-Braga, P. C., 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=Feeder-free+derivation+of+induced+pluripotent+stem+cells+from+human+immature+dental+pulp+stem+cells.">Feeder-free derivation of induced pluripotent stem cells from human immature dental pulp stem cells.</a> Cell Transplant. 20 (11-12), 1707-1719 (2011).</li><li>Haase, A., 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+induced+pluripotent+stem+cells+from+human+cord+blood.">Generation of induced pluripotent stem cells from human cord blood.</a> Cell. Stem Cell. 5 (4), 434-441 (2009).</li><li>Dowey, S. N., Huang, X., Chou, B. K., Ye, Z., Cheng, L. <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+integration-free+human+induced+pluripotent+stem+cells+from+postnatal+blood+mononuclear+cells+by+plasmid+vector+expression.">Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.</a> Nat. Protoc. 7 (11), 2013-2021 (2012).</li><li>Staerk, J., 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=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell. Stem Cell. 7 (1), 20-24 (2010).</li><li>Geti, I., 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=A+practical+and+efficient+cellular+substrate+for+the+generation+of+induced+pluripotent+stem+cells+from+adults:+blood-derived+endothelial+progenitor+cells.">A practical and efficient cellular substrate for the generation of induced pluripotent stem cells from adults: blood-derived endothelial progenitor cells.</a> Stem Cells Transl. Med. 1 (12), 855-865 (2012).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr Protoc Immunol. , Appendix 3-Appendix 3B (2001).</li><li>Hasegawa, K., 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=Comparison+of+reprogramming+efficiency+between+transduction+of+reprogramming+factors,+cell-cell+fusion,+and+cytoplast+fusion.">Comparison of reprogramming efficiency between transduction of reprogramming factors, cell-cell fusion, and cytoplast fusion.</a> Stem Cells. 28 (8), 1338-1348 (2010).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> J Vis Exp. (45), 2565 (2010).</li><li>Fleige, S., Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+integrity+and+the+effect+on+the+real-time+qRT-PCR+performance.">RNA integrity and the effect on the real-time qRT-PCR performance.</a> Mol Aspects Med. 27 (2-3), 126-129 (2006).</li><li>Meraviglia, V., 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=Human+chorionic+villus+mesenchymal+stromal+cells+reveal+strong+endothelial+conversion+properties.">Human chorionic villus mesenchymal stromal cells reveal strong endothelial conversion properties.</a> Differentiation. 83 (5), 260-270 (2012).</li><li>Caspersson, T., Zech, L., Johansson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+human+metaphase+chromosome+set+by+aid+of+DNA-binding+fluorescent+agents.">Analysis of human metaphase chromosome set by aid of DNA-binding fluorescent agents.</a> Exp Cell Res. 253 (2), 302-304 (1999).</li><li>Alikani, M., Munne, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviable+human+pre-implantation+embryos+as+a+source+of+stem+cells+for+research+and+potential+therapy.">Nonviable human pre-implantation embryos as a source of stem cells for research and potential therapy.</a> Stem Cell. Rev. 1 (4), 337-343 (2005).</li><li>Tropel, P., 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=High-efficiency+derivation+of+human+embryonic+stem+cell+lines+following+pre-implantation+genetic+diagnosis.">High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis.</a> In Vitro Cell. Dev. Biol. Anim. 46 (3-4), 376-385 (2010).</li><li>Hanna, J., 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=Direct+cell+reprogramming+is+a+stochastic+process+amenable+to+acceleration.">Direct cell reprogramming is a stochastic process amenable to acceleration.</a> Nature. 462 (72-73), 595-601 (2009).</li><li>Li, H., 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=The+Ink4/Arf+locus+is+a+barrier+for+iPS+cell+reprogramming.">The Ink4/Arf locus is a barrier for iPS cell reprogramming.</a> Nature. 460 (7259), 1136-1139 (2009).</li><li>Kim, K., 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=Epigenetic+memory+in+induced+pluripotent+stem+cells.">Epigenetic memory in induced pluripotent stem cells.</a> Nature. 467 (7313), 285-290 (2010).</li><li>Lindner, S. E., Sugden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasmid+replicon+of+Epstein-Barr+virus:+mechanistic+insights+into+efficient,+licensed,+extrachromosomal+replication+in+human+cells.">The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells.</a> Plasmid. 58 (1), 1-12 (2007).</li></ol>

In passato, l&#39;unico modo per ottenere cellule staminali pluripotenti umane che trasportano una particolare mutazione genetica è stato quello di reclutare genitori sottoposti a diagnosi genetica pre-impianto e generare cellule staminali embrionali da loro blastocisti scartate 31,32. Utilizzando un approccio riprogrammazione, i ricercatori possono ora generare iPSCs di pazienti portatori di praticamente qualsiasi genotipo. La possibilità di partire da una linea cellulare paziente-specifica ed una fonte fa...

Nel 2006, il gruppo di Shinya Yamanaka 1 ha dimostrato per la prima volta che le cellule somatiche di topi adulti e gli esseri umani possono essere convertiti in uno stato pluripotente dall&#39;espressione ectopica di quattro fattori di riprogrammazione (ottobre-4, Sox-2, KLF-4, e c-Myc), generando le cosiddette cellule staminali pluripotenti indotte (iPSCs) 2. IPSCs paziente-specifiche sono molto simili a cellule staminali embrionali umane (hESC) in termini di morfologia, la proliferazione e la capacità di differenziarsi in tipi di cellule germinali (tre mesoderma, endoderma ed ectoderma), mentre mancano le preoccupazioni etiche legate all&#39;uso di hESC e bypass possibile rigetto immunitario 3. Così, iPSCs appaiono come una delle più importanti fonti di cellule paziente-specifiche per la ricerca di base, lo screening di stupefacenti, modelli di malattia, la valutazione della tossicità, e fini di medicina rigenerativa 4. Diversi approcci sono stati utilizzati per la generazione iPSC: vettori virali integrazione(Retrovirus 5, lentivirus 6), virale non integrando vettori (adenovirus 7), Sendai-virus 8, BAC trasposoni 9, vettori episomiale 10, proteine ​​11 o consegna RNA 12. Sebbene l&#39;uso di metodi di virus-mediata può portare ad alta efficienza riprogrammazione, vettori virali integrano nel genoma delle cellule ospiti e quindi potenziale casuale mutagenesi inserzionale, alterazione permanente di espressione genica, e la riattivazione di transgeni tacere durante la differenziazione non può essere esclusa 13. Per rendere più sicuro iPSCs per la medicina rigenerativa, sono stati compiuti sforzi per derivare iPSCs senza l&#39;integrazione di DNA esogeno in genomi cellulari. Sebbene siano stati sviluppati vettori virali soggetti ad accisa e trasposoni, è ancora chiaro se sequenze vettore brevi, che inevitabilmente rimangono nelle cellule trasdotte dopo escissione e transposase espressione, potrebbero indurre alterazioni nella cellalare funzionano 13. Nonostante la sua alta efficienza riprogrammazione, virus Sendai rappresenta un approccio costoso e raggiungere, attraverso problemi di licenza con la società che ha sviluppato questo sistema di avere il potenziale per limitarne l&#39;applicazione negli studi traslazionali. Inoltre, la necessità di introduzione diretta di proteine ​​e RNA richiede consegna multiplo di riprogrammazione molecole con le limitazioni tecniche insite questo introduce, e l&#39;efficienza complessiva riprogrammazione è molto bassa 14. Di nota, metodi virali libere e non integrare convenienti basate sull&#39;uso di plasmidi episomiale sono stati riportati con successo per la riprogrammazione dei fibroblasti cutanei 15. In particolare, in questo lavoro abbiamo deciso di utilizzare commerciali plasmidi episomiale senza integrazione disponibili, come riportato in precedenza 10,15. Fino ad oggi, fibroblasti cutanei rappresentano il più popolare tipo di cellula donatrice 5. Tuttavia, altre fonti di cellule sono stati successfully riprogrammato in iPSCs compresi cheratinociti 16, del midollo osseo cellule staminali mesenchimali 17, cellule stromali adipose 18, follicoli piliferi 19, e le cellule della polpa dentale 20. L&#39;isolamento di queste cellule richiede procedure chirurgiche, e diverse settimane sono necessari per l&#39;espansione delle cellule in vitro allo scopo di stabilire una coltura cellulare primaria. In questa luce, la selezione del tipo di cellula di partenza è critico ed è altrettanto importante essere in grado di produrre iPSCs dai tessuti facilmente accessibili e meno invasive come sangue. Entrambe le cellule mononucleate del sangue del cordone (CBMNCs) 21,22 e cellule mononucleari di sangue periferico (PBMNCs) 14,22-24 rappresentano idonee fonti di cellule per la derivazione di iPSCs. Sebbene l&#39;efficienza di adulti PBMNC riprogrammazione è 20-50 volte inferiore a quella del CBMNCs 22, rimangono il tipo di cella più conveniente per scopo campionamento. InInfatti, campionamento PBMNC ha il vantaggio di essere minimamente invasiva, e in aggiunta, queste cellule non richiedono vasta espansione in vitro prima degli esperimenti di riprogrammazione. Fino ad oggi, i protocolli differenti hanno riferito che PBMNCs dopo la separazione gradiente di densità possono essere congelati e scongelati giorni a diversi mesi dopo il congelamento e ampliato per alcuni giorni prima della riprogrammazione in iPSCs 22,23. Tuttavia, per quanto a nostra conoscenza segnalazioni hanno descritto una riprogrammazione della PBMNCs da buffy coats congelati. È importante sottolineare che, buffy coats congelati raccolti senza separazione gradiente di densità rappresentano i campioni di sangue più comuni memorizzati nella biobanche di grandi dimensioni provenienti da studi di popolazione, rappresentando così una piscina facilmente accessibile di materiale per la produzione di IPSC che evita ulteriormente la raccolta dei campioni. Qui riportiamo per la prima volta la generazione di iPSCs-virali privi di buffy coats congelati umani, sulla base di un protocollo precedentemente descritto 22. InInoltre, sono stati generati da iPSCs PBMNCs congelati ottenuti dopo la separazione gradiente di densità, come un protocollo di controllo per i risultati PBMNC gradiente di densità non depurati.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Citrate buffered Venosafe Plastic Tube</td>
			<td>Terumo</td>
			<td>VF-054SBCS07</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>60339</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium powder</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td>0.5 M solution</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Premium&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-5442-02</td>
			<td>Polysucrose solution for density gradient centrifugation</td>
		</tr>
		<tr>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM)&#160;</td>
			<td>Gibco</td>
			<td>21056-023</td>
			<td>No phenol red</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12</td>
			<td>Mediatech</td>
			<td>10-080-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-Ethanolamine (ITS -X)</td>
			<td>Gibco</td>
			<td>51500-056&#160;</td>
			<td>1X (Stock: 100X)</td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td>1X (Stock: 100X)</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Thioglycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M6145</td>
			<td>Final Concentration at 200 &#181;M</td>
		</tr>
		<tr>
			<td>Recombinant Human Stem Cell Factor (SCF)</td>
			<td>PeproTech</td>
			<td>300-07</td>
			<td>100 ng/ml (Stock:100 &#181;g/ml)&#160;</td>
		</tr>
		<tr>
			<td>Recombinant Human Interleukin-3 (IL-3)</td>
			<td>PeproTech</td>
			<td>200-03</td>
			<td>10 ng/ml (Stock: 10 &#181;g/ml)</td>
		</tr>
		<tr>
			<td>Recombinant Human Insulin-like Growth Factor (IGF-1)</td>
			<td>PeproTech</td>
			<td>100-11</td>
			<td>40 ng/ml (Stock: 40 &#181;g/ml)</td>
		</tr>
		<tr>
			<td>Recombinant Human Erythropoietin (EPO)</td>
			<td>R&#38;D Systems&#160;</td>
			<td>287-TC-500</td>
			<td>2 U/ml (Stock: 50 U/ml)</td>
		</tr>
		<tr>
			<td>Dexamethasone&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D2915</td>
			<td>1 &#956;M (Stock: 1 mM)</td>
		</tr>
		<tr>
			<td>Human Holo-Transferrin</td>
			<td>R&#38;D Systems&#160;</td>
			<td>2914-HT</td>
			<td>100 &#956;g/ml (Stock: 20 mg/ml)</td>
		</tr>
		<tr>
			<td>Amniomax II</td>
			<td>Gibco</td>
			<td>11269016</td>
			<td>Medium for cytogenetic analysis</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>StemCell Technologies</td>
			<td>5850</td>
			<td>Medium for iPSC feeder-free culture</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol&#160;</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td>0.1 mM (Stock: 50 mM)</td>
		</tr>
		<tr>
			<td>Sodium Butyrate</td>
			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td>0.25 mM (Stock: 0.5 M)</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF basic, 145 aa&#160;</td>
			<td>R&#38;D Systems&#160;</td>
			<td>4114-TC</td>
			<td>10 ng/ml (Stock: 10 &#181;g/ml)</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503&#160;</td>
			<td>10 &#181;M (Stock: 10 mM)</td>
		</tr>
		<tr>
			<td>Fetal Defined Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH 30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax 0.1% Gelatin Solution</td>
			<td>Merck-Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td>1 mg/ml (Stock: 10 mg/ml)</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>PAA Laboratories GmbH</td>
			<td>L11-007</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Mouse Embryonic Fibroblast (CF1)</td>
			<td>Global Stem</td>
			<td>GSC-6201G</td>
			<td>1*106 cells/6 well plate</td>
		</tr>
		<tr>
			<td>Plasmid pCXLE-hOCT3/4-shp53-F</td>
			<td>Addgene&#160;</td>
			<td>27077</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Plasmid pCXLE-hSK</td>
			<td>Addgene&#160;</td>
			<td>27078</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Plasmid pCXLE-hUL</td>
			<td>Addgene&#160;</td>
			<td>27080</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Plasmid pCXLE-EGFP</td>
			<td>Addgene&#160;</td>
			<td>27082</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Staining Kit</td>
			<td>Stemgent</td>
			<td>00-0009</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Stage-Specific Embryonic Antigen-4 (SSEA-4) Antibody</td>
			<td>Merck-Millipore</td>
			<td>MAB4304</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Anti-TRA-1-60 Antibody</td>
			<td>Merck-Millipore</td>
			<td>MAB4360</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Anti-TRA-1-81 Antibody</td>
			<td>Merck-Millipore</td>
			<td>MAB4381</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Anti-Oct-3/4 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9081</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Anti-Nanog Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-33759</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Anti-Troponin I Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-15368</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Anti-&#945;-Actinin (Sarcomeric) Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A7732</td>
			<td>1/250</td>
		</tr>
		<tr>
			<td>Neuronal Class III &#223;-Tubulin (TUJ1) Antibody</td>
			<td>Covance Research Products Inc&#160;</td>
			<td>MMS-435P-100</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Anti-Tyrosine Hydroxylase (TH) Antibody</td>
			<td>Calbiochem</td>
			<td>657012</td>
			<td>1/200</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse&#160;</td>
			<td>Molecular Probes</td>
			<td>A-11029</td>
			<td>1/1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Goat Anti-Rabbit</td>
			<td>Molecular Probes</td>
			<td>A-21429</td>
			<td>1/1000</td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Cell Culture 6-well plate</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol Reagent</td>
			<td>Ambion</td>
			<td>15596-018&#160;</td>
			<td>reagent for RNA extraction</td>
		</tr>
		<tr>
			<td>SuperScript VILO cDNA Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>11754050</td>
			<td>Reverse transcriptase kit</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Bio-Rad</td>
			<td>172-5124</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>185-5195</td>
			<td></td>
		</tr>
		<tr>
			<td>Experion Automated Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td>700-7000</td>
			<td>Instrument to check RNA integrity</td>
		</tr>
		<tr>
			<td>Experion RNA Highsense Analysis kit</td>
			<td>Bio-Rad</td>
			<td>7007105</td>
			<td>Reagent kit to check RNA integrity</td>
		</tr>
		<tr>
			<td>Dissecting microscope (SteREO Discovery V12 )</td>
			<td>Zeiss</td>
			<td>495007</td>
			<td></td>
		</tr>
		<tr>
			<td>NeonTransfection System 100 &#181;L Kit</td>
			<td>Invitrogen</td>
			<td>MPK10025</td>
			<td>Reagent kit for electroporation</td>
		</tr>
		<tr>
			<td>Neon Transfection System</td>
			<td>Invitrogen</td>
			<td>MPK5000</td>
			<td>Instrument used for electroporation</td>
		</tr>
		<tr>
			<td>NanoDrop UV/Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>Instrument for DNA/RNA quantification</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>Plasmid purification kit</td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from frozen buffy coats using non-integrating episomal plasmids

Cellule somatiche possono essere riprogrammate in cellule staminali pluripotenti indotte (iPSCs) forzando l&#39;espressione di quattro fattori di trascrizione (ottobre-4, Sox-2, KLF-4, e c-Myc), tipicamente espressa da cellule staminali embrionali umane (hESC) . A causa della loro somiglianza con hESC, iPSCs sono diventati uno strumento importante per il potenziale di medicina rigenerativa paziente-specifici, evitando questioni etiche connesse con hESC. Al fine di ottenere cellule adatte per l&#39;applicazione clinica, iPSCs senza transgene devono essere generati per evitare transgene riattivazione, alterata espressione genica e differenziazione sbagliata. Inoltre, un metodo di riprogrammazione molto efficiente e conveniente è necessario ricavare iPSCs sufficienti a fini terapeutici. Tenuto conto di questa esigenza, un approccio efficiente non integrando episomiale plasmide è la scelta preferibile per IPSC derivazione. Attualmente il tipo di cella più comune utilizzato per scopi di riprogrammazione sono fibroblasti, l&#39;isolamento dei quali richiede biopsia tissutale, un iprocedura chirurgica nvasive per il paziente. Pertanto, sangue periferico umano rappresenta il tessuto più accessibile e meno invasivo per la generazione IPSC. In questo studio, un protocollo virale senza costo-efficacia e l&#39;utilizzo non integrano plasmidi episomiale è segnalato per la generazione di iPSCs dal periferico cellule umane mononucleate del sangue (PBMNCs) ottenute da buffy coats congelati dopo centrifugazione del sangue intero e senza la densità di separazione gradiente.

In questo studio un protocollo semplice ed efficace per la generazione di iPSCs-virali libero da riprogrammazione della PBMNCs isolati da buffy coat congelati dopo centrifugazione del sangue intero e confronto con riprogrammazione della PBMNCs ottenute dopo la separazione gradiente di densità è riportato. La figura 1A mostra una rappresentazione schematica di il protocollo dettagliato. Dopo lo scongelamento, i PBMNCs isolato, mostrando una tipica forma arrotondata, vengono espanse in specifico terreno...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids at JoVE.com

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids (Video) | JoVE

Cellule staminali pluripotenti indotte (iPSCs) rappresentano una fonte di tessuti paziente-specifici per le applicazioni cliniche e di ricerca di base. Qui vi presentiamo un protocollo dettagliato per riprogrammare le cellule umane mononucleari di sangue periferico (PBMNCs) ottenute da buffy coats congelati in iPSCs-virali gratuito utilizzando non integrano plasmidi episomiale.

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, ...

Research

JoVE Journal

Developmental Biology

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

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes (Video) | JoVE

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 ...

Ricerca

Biologia dello sviluppo

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

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions (Video) | JoVE

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

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients (Video) | JoVE

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

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes (Video) | JoVE

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

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation (Video) | JoVE

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

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors (Video) | JoVE

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

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells (Video) | JoVE

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 ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells (Video) | JoVE

Formazione di pellet condensato da cellule staminali pluripotenti indotte dal sangue

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells (Video) | JoVE

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. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells (Video) | JoVE

Generazione di pelle 3D Organoid dal cordone emoderivati indotta da cellule staminali pluripotenti indotte

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...