Name: generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions
Uploaded: 2015-11-11T15:00:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

Vi takker Yoshiko Miyake, Sayaka Kanaami, Chihana Fujita, Miho Yamaguchi, Natsuko Henmi, og Rei Ohno fra Keio University School of Medicine til teknisk bistand. Dette arbejde blev delvist finansieret af en R &amp; D Systems støtteprogram til at fremskynde den praktiske anvendelse af sundheds- forskningsresultater, og Highway Program for realiseringen af ​​regenerativ medicin.

1. Klargør Aktiverede humane T-celler <ol><li> Saml 10 ml hepariniserede fuldblod fra en donor ved venepunktur. <ol><li> Indhente informeret samtykke fra donorer forud for venepunktur. Venepunktur skal udføres af kvalificeret og uddannet personale. </li><li> Håndtag opnået blodprøver med sterilt udstyr og følgende relevante retningslinjer biosikkerhed. </li></ol></li><li> Fortynd 10 ml hepariniserede helblod med 10 ml D-PBS (-). </li><li> Sæt 15 ml Ficoll-opløsning (Ficoll-Paque Premium) i en ny 50 ml konisk rør. </li><li> Lag 20-ml blod opløsning fremstillet i trin 1.2 på Ficoll opløsning i trin 1.3. Anvendelse af en elektrisk pipette, lad blod opløsning fremstillet i trin 1.2 løb langsomt langs med indersiden af ​​røret. Pas på ikke at blande blodopløsningen og Ficoll løsning. </li><li> Der centrifugeres ved 400 x g i 30 minutter ved stuetemperatur. Sæt centrifugal deceleration hastighed til &quot;langsom&quot;. Bemærk: Efter centrifugering en hvid tyndlag opstår mellem en øvre mælkeagtig plasma lag og et nedre klart Ficoll lag. Denne hvide tyndt lag indeholder mononukleare celler. </li><li> Overfør mononukleære cellelag i et nyt 50-ml konisk rør med en 1-ml pipette. Pas på ikke at medtage Ficoll løsning. </li><li> Tilføj D-PBS (-) til mononukleære celler til et samlet volumen på 10 ml, og der centrifugeres ved 200 x g i 5 minutter ved stuetemperatur. </li><li> Kassér supernatanten. Der tilsættes 10 ml D-PBS (-) til mononukleære celler, og der centrifugeres ved 200 x g i 5 minutter ved stuetemperatur. </li><li> Supernatanten fjernes, tilsættes 1 ml KBM502 medium til de indsamlede mononukleære celler, og tæl cellerne under anvendelse af et hæmocytometer. Over 5 × 10 6 mononukleære celler kan fås fra 10 ml hepariniserede fuldblod med passende adskillelse hjælp Ficoll. </li><li> Forbered et anti-humant CD3-antistof ved en koncentration på 10 ug / ml i D-PBS (-). Tilsæt anti-humant CD3-antistof-opløsning til en 6-brønds plade, sættetid brændingenes hver brønd, og inkuber fadet ved 37 ° C i en 5% CO2-inkubator i mindst 30 minutter. </li><li> Fjern anti-humant CD3-antistof-opløsning fra hver brønd og vask en gang med D-PBS (-) lige før podning af cellerne. </li><li> Seed mononukleære celler, som blev fremstillet i trin 1.9 med en tæthed på 2,5 x 10 5 celler / cm2 på den anti-humant CD3-antistof-coatede plader med 6 brønde i KBM502 medium. Der inkuberes ved 37 ° C i en 5% CO 2-inkubator i 3-7 dage uden udskiftning af mediet, indtil T-cellerne flyder sammen. T-celler i denne periode er begge suspenderede og vedhængende celler. </li></ol> 2. inficere humane T-celler med SeV Vektorer <ol><li> Efter 3-7 dages dyrkning, indsamle de aktiverede T-celler med eksisterende medium ved pipettering. Overføre dem til en 15 ml konisk rør og centrifugeres ved 200 x g i 5 minutter ved stuetemperatur. </li><li> Fjern supernatanten og tilsæt 1 ml frisk KBM502 medium. Tæl cellerne og pladen 1.5 x 10 6 celler (i 2 ml frisk KBM502 medium) i hver brønd på et anti-CD3-antistof-coatede plade med 6 brønde. </li><li> Optø SEV løsninger OCT3 / 4-SeV / TSΔF, SOX2-SeV / TSΔF, KLF4-SeV / TSΔF, og c-MYC (HNL) -SeV / TS15ΔF på is. </li><li> Tilføj SEV opløsninger indeholdende OCT3 / 4-SeV / TSΔF, SOX2-SeV / TSΔF, KLF4-SeV / TSΔF og c-MYC (HNL) -SeV / TS15ΔF individuelt til brøndene, hver ved en infektionsmultiplicitet (MOI) på 10. Der inkuberes ved 37 ° C i en 5% CO 2 inkubator i yderligere 24 timer. </li></ol> 3. Fjern SeV Vektorer fra humane T-celler <ol><li> 24 timer efter infektion, indsamle de inficerede celler med en pipette. Hvis der er adhærerende celler, kan de nemt adskilles pipettering dem flere gange op og ned. Overføre dem til en 15 ml konisk rør, og der centrifugeres ved 200 x g i 5 minutter ved stuetemperatur. </li><li> Fjern supernatanten indeholdende SEV vektorer og tilsæt 2 ml frisk KBM502 medium. Udpladning celler i each af brøndene. Der inkuberes ved 37 ° C i en 5% CO 2 inkubator i yderligere 24 timer. </li></ol> 4. reseed Celler på en Matrigel Layer <ol><li> Basalmembranmatrix opløsning ved optøning matrixgel forberede (se venligst Materialer List for specifikt mærke anvendt i denne undersøgelse) ved 4 ° C og opløse 0,2 ml i 10 ml DMEM / F12-medium. Bemærk: Matrix gel (se venligst Materialer List for specifikt mærke anvendt i denne undersøgelse) skal tø O / N ved 4 ° C for at være helt optøet. Holde den på is indtil lige før anvendelse. </li><li> Pladen 5 ml basalmembranmatrix opløsning pr skål i 100 mm skåle og henstår ved stuetemperatur i mindst 30 minutter før såning celler. Der er behov for mindst to 100-mm-retter til et eksperiment. </li><li> Saml SeV-inficerede celler ved pipettering, overføre dem til en 15 ml konisk rør, og der centrifugeres ved 200 x g i 5 minutter ved stuetemperatur. </li><li> Fjern supernatanten og tilsæt 1 ml frisk KBM502 medium. Tæl antallet af ceLLS anvendelse af et hæmocytometer, fjerne basalmembranmatrix løsning fra 100 mm skåle fremstillet i trin 4.2 og pladen 1 × 10 5 og 1 x 10 6 celler (i 10 ml frisk mTeSR medium) på en 100 mm basalmembranmatrix-overtrukne retter. Brug til sidst pladen, hvor kolonier nemt plukkes. </li><li> Inkubér fadet ved 37 ° C i en 5% CO 2 inkubator O / N. </li><li> Skift mediet til 10 ml kød mTeSR medium hver anden dag. Bemærk: Ca. 20-30 dage efter infektion, kolonier, der nøje ligner embryonale stamceller (EKSF) vises. </li></ol> 5. Expand T celleafledt iPSCs <ol><li> Basalmembranmatrix opløsning ved optøning matrixgel forberede (se venligst Materialer List for specifikt mærke anvendt i denne undersøgelse) ved 4 ° C og opløse 0,2 ml i 10 ml DMEM / F12-medium. </li><li> Plade 1 ml af basalmembranmatrix opløsning per brønd i en 6-brønds plade og henstår ved stuetemperatur imindst 30 min før plukke kolonier. </li><li> Fyld en plade med 96 brønde med 20 pi mTeSR medium per brønd. </li><li> Vælg kolonier, der ligner økonomiske og sociale råd under stereomikroskop ved hjælp af en 20 pi pipette. Bemærk: Kolonierne af økonomiske og sociale råd iPSCs er runde og flade som vist i figur 1B. </li><li> Overfør udvalgte kolonier i 96-brønds plade fyldt med mTeSR medium i trin 5.3. </li><li> Tilsæt 200 pi mTeSR medium til hver brønd og omhyggeligt pipetteres op og ned for at bryde kolonien i små klumper under stereomikroskop ved anvendelse af en 200 pi pipette. Fjern basalmembranmatrix løsning fra brønden forberedt på 5,2 og sætte hver koloni i en brønd af basalmembranen matrix-coatede plade med 6 brønde med 2 ml mTeSR medium per brønd. </li><li> Skift mediet til 2 ml kød mTeSR medium hver anden dag. </li></ol> 6. Oprethold T celleafledt iPSCs T-celle-afledte iPSCs kan opretholdesog gemmes ved hjælp af de samme teknikker som for humane iPSCs og menneskelige økonomiske og sociale råd. Når IPSC kolonier vokser op, udvide dem til større retter ved at gentage den samme procedure. <ol><li> Skift mTeSR medium hver 1-2 dage. </li><li> Når kolonierne bliver sammenflydende hver 5-7 dage, passage cellerne under anvendelse dissocieringsopløsning for humane iPSCs og menneskelige ESC i overensstemmelse med producentens anvisninger. </li></ol>

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We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

iPSCs har tiltrukket stor opmærksomhed som en banebrydende kilde til celler til regenerativ medicin 1-3. Til dato er der rapporteret diverse metoder til at generere iPSCs 4,5. Blandt disse har iPSCs genereret fra humane T-celler været af særlig interesse på grund af de mindre invasiv metode til celleprøveudtagningsproceduren 6-8. Derudover iPSCs afledt af T-celler indeholder omlejringer af T-cellereceptoren (TCR) genet og er således en kilde til antigen-specifikke T-celler 9,10. Derfor generere T-celle afledt iPSCs sikkert er nyttig til forløber regenerativ medicin. Denne metode er baseret på princippet om at reducere risikoen for uforudsigelige patogenicitet. For sikker klinisk anvendelse af iPSCs er det vigtigt at reducere risikoen for eksponering for patogener 11. Tidligere i mange dyrkningssystemer af pluripotente stamceller, har kalvefosterserum og fødeceller blevet anvendt som væsentlige reagenss 12. Dog er udtagelse af begge disse reagenser fra dyrkningssystemet er at foretrække for iPSC generation til at reducere risikoen for uforudsigelige patogenicitet. Desuden er denne fremgangsmåde har den fordel, at man undgår invasive celleprøveudtagningsproceduren fra patienter og besværlig fremstilling af fødeceller. Fordi T-celle-afledte iPSCs allerede er blevet anvendt med succes i sygdom forskning 13,14, er denne fremgangsmåde også anvendelig og nyttig til frembringelse af sygdomsspecifikke iPSCs fra patienter. Blandt T-celle-omprogrammering metoder, ved hjælp af Sendai-virus (SeV) vektor som et gen køretøj er en metode, der kan generere iPSCs med høj effektivitet 7,16. Desuden, fordi SeV er en enkeltstrenget RNA-virus og behøver ikke en DNA fase for replikation, dets anvendelse i iPSC generation undgår at bryde værtsgenomet 17-19. Derfor har vi etableret protokoller til generering iPSCs fra humane perifere T-celler i serum-frEE og fødefri betingelser ved anvendelse af en kombination af matrigel, mTeSR medium og SeV-vektorer.

<table border="0">
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified NA/LE mouse anti-human CD3</td>
			<td>BD Pharmingen</td>
			<td>555336</td>
			<td></td>
		</tr>
		<tr>
			<td>KBM502 medium</td>
			<td>KOHJIN BIO</td>
			<td>16025020</td>
			<td>Warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4&#8451; overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEM CELL</td>
			<td>5850</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr>
			<td>Dissociation Solution</td>
			<td>ReproCELL</td>
			<td>RCHETP002</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(&#8211;)</td>
			<td>Wako</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>SeV Vector kit CytoTune-iPS ver.1.0</td>
			<td>DNAVEC</td>
			<td>DV-0303c</td>
			<td>Thaw on ice before use</td>
		</tr>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353078</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>227261</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions

For nylig har tiltrukket sig opmærksomhed iPSCs som en ny kilde af celler til regenerative terapier. Selv om den oprindelige fremgangsmåde til generering iPSCs påberåbte dermale fibroblaster opnået ved invasiv biopsi og retroviral genomisk indsættelse af transgener, har der været mange forsøg på at undgå disse ulemper. Humane perifere T-celler er en unik cellekilde til generering iPSCs. iPSCs afledt af T-celler indeholder omlejringer af T-cellereceptoren (TCR) gener og er en kilde til antigen-specifikke T-celler. Derudover T-cellereceptor omlejring i genomet har potentiale til at mærke individuelle cellelinier og skelne mellem transplanterede og donorceller. For sikker klinisk anvendelse af iPSCs, er det vigtigt at minimere risikoen for at udsætte nyligt genererede iPSCs for skadelige stoffer. Selv kalvefosterserum og fødeceller har været af afgørende betydning for pluripotente stamcelle kultur, foretrækkes det at fjerne dem fra dyrkningssystemet at mindske risikoenaf uforudsigelige patogenicitet. For at løse dette, har vi etableret en protokol til generering iPSCs fra humane perifere T-celler under anvendelse af Sendai-virus for at reducere risikoen for at udsætte iPSCs udefinerede patogener. Selvom håndtering Sendai-virus kræver udstyr med passende level-Sendai virus inficerer aktiverede T-celler uden indføring genom, men med høj effektivitet. I denne protokol, viser vi dannelsen af ​​iPSCs fra humane perifere T-celler i fødefri betingelser under anvendelse af en kombination af aktiveret T-celle-kultur og Sendai-virus.

<html> Ved hjælp af denne protokol, brugerne er i stand til at generere iPSCs fra humane perifere T-celler stabilt. iPSC generation fra T-celler med SeV og matrigel viste ca. 0,002% -. 0,005% af effektiviteten af celle omprogrammering i mellem flere donor sager og SeV blev ikke påvist efter flere passager 16 Figur 1A viser en skematisk af protokollen til at generere T-celle-afledt iPSCs i feeder-fri betingelser ved anvendelse matrigel og mTeSR medium. Omkring 20-30 dage efter infektion med SeV, er IPSC kolonier genkendes ved deres ESC koloni-morfologi (figur 1B). Immunfluorescensfarvning afslørede udtryk for typiske pluripotente cellemarkører (NANOG, OCT3 / 4, SSEA4, TRA-1-60, og TRA-1-81) i T-celle-afledte iPSCs genereret under feeder-fri betingelser (figur 1C). &quot;Width =&quot; 700 &quot;/&gt; Figur 1. (A): En skematisk af protokollen for omprogrammering T-celler under feeder-fri forhold i denne undersøgelse. (B): En typisk ESC-lignende iPSC koloni på dag 27 efter blodprøvetagning under fødefri betingelser. (C): ALP farvning og immunofluorescensfarvning til pluripotens og overflademarkører (NANOG, OCT3 / 4, SSEA4, TRA-1-60, og TRA-1-81) i T-celle-afledte iPSCs genereret under feeder-fri betingelser. <a href="https://www.jove.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">Venligst klik her for at se en større version af dette tal.</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

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

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.

Generering af induceret pluripotente stamceller fra humane perifere T-celler Brug af Sendai Virus i Feeder-fri Betingelser

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

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