Name: efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus
Uploaded: 2014-04-23T22:00:00
Description: Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus Video at JoVE.com

Vi vil gerne takke medlemmerne af Lee laboratorium til værdifulde diskussioner om manuskriptet. Arbejdet i Lee lab blev støttet af tilskud fra Robertson Investigator Award i New York Stem Cell Foundation og fra Maryland Stem Cell Research Fund (TEDCO).

1.. Udarbejdelse af Cell og Medier (dag 1) <ol><li> Kultur og udvide human fibroblast med DMEM-medium indeholdende 10% FBS. </li><li> Plate humane fibroblaster (figur 1) på en 24-brønds plade ved passende tæthed per brønd på dagen før transduktion. BEMÆRK: Følgende seriefortyndinger anbefales (200K, 100K, 50K, 25K, 12.5K og 6.25K), fordi forskellige celletyper har forskellig tilknytning evne. </li><li> Inkuberes cellerne for en dag mere i et 37 ° C, 5% CO 2 ...

<ol>
	<li>Yu, 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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., 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=Generation+of+mouse+induced+pluripotent+stem+cells+without+viral+vectors.">Generation of mouse induced pluripotent stem cells without viral vectors.</a> Science. 322, 949-953 (2008).</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, 2667-2674 (2009).</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, 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> Nature Methods. 8, 409-412 (2011).</li><li>Yusa, K., Rad, R., Takeda, J., Bradley, A. <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+transgene-free+induced+pluripotent+mouse+stem+cells+by+the+piggyBac+transposon.">Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.</a> Nature Methods. 6, 363-369 (2009).</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, 472-476 (2009).</li><li>Yu, 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=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Okita, K., Ichisaka, T., 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=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Yoshioka, 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=Efficient+Generation+of+Human+iPSCs+by+a+Synthetic+Self-Replicative+RNA.">Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA.</a> Cell Stem Cell. 13, 246-254 (2013).</li><li>Dowey, S. 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Omprogrammering humane somatiske celler til hiPSCs besidder hidtil usete løfter i grundlæggende biologi, personlig medicin og transplantation 12. Tidligere human IPSC generationer krævede DNA-virus, der har integration risiko i værtens genom, som kan skabe uønskede genetiske mutationer, der begrænser yderligere kliniske applikationer såsom lægemiddeludvikling og transplantationscentre terapier 13. Med denne grund har mange undersøgelser blevet rapporteret at generere vektorbaseret og trans...

Humane embryonale stamceller (hESCs) har en kapacitet til selv at forny in vitro og har pluripotency, hvilket kan være potentielt nyttige for sygdom modellering, til drug screening, og for at udvikle cellebaserede behandlingsformer til at behandle skader sygdom og væv. Men hESCs har en begrænsning for celleudskiftning grund af immunologiske, onkologiske og etiske barrierer, og til at studere sygdomsrelaterede gener, kunne sygdomsspecifikke hESCs isoleres gennem præ-implantation genetisk diagnose (PGD) nærmer sig, men det er stadig teknisk udfordrende og embryoet donationer er temmelig sjældne. Disse problemer er relateret til udviklingen i stamcellebiologiske, hvilket har ført til udviklingen af ​​inducerede pluripotente stamceller (hiPSCs). Menneskelige iPSCs er genetisk omprogrammeres fra humane voksne somatiske celler og havnen pluripotente stamceller-lignende funktioner ligner hESCs, hvilket gør dem en nyttig kilde til regenerativ medicin, såsom dtæppe opdagelse, modellering sygdom og celleterapi i patient-specifik måde 1,2. Indtil nu, er der flere metoder til at generere menneskelige iPSCs, herunder virus-medieret (retrovirus og adenovirus) 3 non-virus medieret (BAC system og vektorer transfektionsreagenser) 4 gen transduktioner og protein fremføringssystemer 5-7. Selv om en levering af virus-medierede gener kan sikre et vist niveau af effektivitet, kan virale vektorer forlade genetiske fodaftryk, fordi de integreres i værtskromosomerne at udtrykke omprogrammering gener på en ukontrolleret måde. Selv når viral integration af transkriptionsfaktorer kan aktivere eller inaktivere værtsgener 8, kan det forårsage en uventet genetisk aberration og risikoen for tumorigenese 5,9. På den anden side blev den direkte indføring af proteiner eller RNA i somatiske celler rapporteret, men har nogle ulemper, såsom arbejdskrævende, gentagne transffdeling, og lavt omprogrammering 7,10. Selv episomale og ikke-integrerende adenovirus, adeno-associeret virus og plasmidvektorer stadig er relativt mindre effektive 11. Af disse grunde er det plausibelt at vælge ikke-integration omprogrammering metoder med høj effektivitet af IPSC generation og færre genetiske abnormiteter. I denne undersøgelse, bruger vi en Sendai-virus baseret omprogrammering. Denne metode er kendt for at være ikke-integreret i værtsgenomet og konsekvent producerer menneskelige iPSCs uden transgene integrationer.

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			<td height="21" style="height:21px;width:543px;">SSEA-4</td>
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			<td>Santa Cruz</td>
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			<td height="21" style="height:21px;width:543px;">QuantiTect Reverse Transcription Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td>205313</td>
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			<td height="23" style="height:23px;">PCR Master Mix [2X]</td>
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			<td style="width:119px;">K0171</td>
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			<td height="21" style="height:21px;">Trizol</td>
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			<td>15596018</td>
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			<td height="21" style="height:21px;">picking hood</td>
			<td>NuAire</td>
			<td style="width:119px;">&#160;NU-301&#160;</td>
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			<td height="21" style="height:21px;width:543px;">dissecting scope&#160;</td>
			<td style="width:195px;">Nikon</td>
			<td>SMZ745</td>
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efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

For et par år siden, etablering af menneskelige inducerede pluripotente stamceller (iPSCs) indvarslede en ny æra inden for biomedicin. Potentielle anvendelser af menneskelige iPSCs omfatter modellering patogenesen af ​​genetiske sygdomme hos mennesker, autolog celleterapi efter gen korrektion, og personlig drug screening ved at give en kilde til patient-specifikke og symptom relevante celler. Der er imidlertid flere forhindringer at overvinde, såsom at fjerne de resterende omprogrammering faktor transgenekspression efter humane iPSCs produktion. Endnu vigtigere er, kan resterende transgen ekspression i udifferentierede humane iPSCs hæmmer ordentlig differentieringer og misguide fortolkningen af sygdommen relevante in vitro-fænotyper. Med denne grund har integration-fri og / eller transgene fri humane iPSCs blevet udviklet ved hjælp af flere metoder, såsom adenovirus, den piggyBac system minicircle vektor, episomale vektorer, direkte protein levering og syntetiseres mRNA. Men effektiviteten af ​​reprogramming hjælp integration-fri metoder er ganske lav i de fleste tilfælde. Her præsenterer vi en metode til at isolere menneskelige iPSCs ved hjælp af Sendai-virus (RNA-virus) baseret omprogrammering system. Denne omprogrammering metode viser konsistente resultater og høj effektivitet i omkostningseffektiv måde.

Normalt inficerede fibroblaster ikke udviser nogen morfologiske ændringer i flere dage efter Sendai virus transduktion, men fem dage senere, de begynder at have forskellige former (figur 1). Som beskrevet i en højre panel i figur 1, behøver cellerne ikke har typisk fibroblast morfologi mere. De har en rund form og større kerne end cytoplasma. Selv når transduktion udføres i 80% cellulær konfluens, ser det ud som de er mindre sammenflydende i brønden efter at de begynder at ompro...

Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus Video at JoVE.com

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus Video (Video) | JoVE

Her præsenterer vi vores etablerede metode til at omprogrammere humane somatiske celler i transgene-fri menneskelige iPSCs med Sendai-virus, som viser ensartet resultat og øget effektivitet.

Effektiv Generation Menneskelige induceret pluripotente stamceller fra humane somatiske celler med Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

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