Name: integration free derivation of human induced pluripotent stem cells using laminin 521 matrix
Uploaded: 2017-07-07T15:00:00
Description: Watch this Scientific Journal Video about Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix at JoVE.com

Questo lavoro è stato sostenuto da agenti di finanziamento SSF (B13-0074) e VINNOVA (2016-04134).

La raccolta del materiale del paziente e la derivazione delle cellule hiPS è approvata dal Consiglio di revisione dell&#39;etica, Stoccolma, 28 marzo 2012, Numero di registrazione: 2012 / 208-31 / 3. Le fasi della coltura cellulare devono essere eseguite in armadi di biosicurezza, salvo diversa indicazione. Praticare sempre tecniche di manipolazione sterili quando si lavora con le cellule. Lasciare che i supporti, le lastre e i reagenti raggiungano la temperatura ambiente prima di iniziare. Incubare le cellule a 37 ᵒ...

<ol>
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B., Okamoto, S., Xiang, P., Takahashi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration-free+induced+pluripotent+stem+cells+derived+from+retinitis+pigmentosa+patient+for+disease+modeling.">Integration-free induced pluripotent stem cells derived from retinitis pigmentosa patient for disease modeling.</a> Stem Cells Transl Med. 1 (6), 503-509 (2012).</li><li>Zhou, H., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+induced+pluripotent+stem+cell+technology.">Evolution of induced pluripotent stem cell technology.</a> Curr Opin Hematol. 17 (4), 276-280 (2010).</li><li>Thomson, J. 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>Hovatta, O., 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+culture+system+using+human+foreskin+fibroblasts+as+feeder+cells+allows+production+of+human+embryonic+stem+cells.">A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.</a> Hum Reprod. 18 (7), 1404-1409 (2003).</li><li>Ludwig, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-independent+culture+of+human+embryonic+stem+cells.">Feeder-independent culture of human embryonic stem cells.</a> Nat Methods. 3 (8), 637-646 (2006).</li><li>Rodin, S., 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=Long-term+self-renewal+of+human+pluripotent+stem+cells+on+human+recombinant+laminin-511.">Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.</a> Nat Biotech. 28 (6), 611-615 (2010).</li><li>Chen, 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=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nat Meth. 8 (5), 424-429 (2011).</li><li>Rodin, S., Antonsson, L., Hovatta, O., Tryggvason, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer+culturing+and+cloning+of+human+pluripotent+stem+cells+on+laminin-521-based+matrices+under+xeno-free+and+chemically+defined+conditions.">Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.</a> Nat Protoc. 9 (10), 2354-2368 (2014).</li><li>Rodin, S., 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=Clonal+culturing+of+human+embryonic+stem+cells+on+laminin-521/E-cadherin+matrix+in+defined+and+xeno-free+environment.">Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.</a> Nat Commun. 5, 3195 (2014).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proc Jpn Acad Ser B Phys Biol Sci. 85 (8), 348-362 (2009).</li><li>Warren, L., 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=Highly+Efficient+Reprogramming+to+Pluripotency+and+Directed+Differentiation+of+Human+Cells+with+Synthetic+Modified+mRNA.">Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA.</a> Cell Stem Cell. 7 (5), 618-630 (2010).</li><li>Unger, C., Skottman, H., Blomberg, P., Dilber, M. S., Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+manufacturing+practice+and+clinical-grade+human+embryonic+stem+cell+lines.">Good manufacturing practice and clinical-grade human embryonic stem cell lines.</a> Hum Mol Genet. 17 (R1), R48-R53 (2008).</li><li>Martin, M. J., Muotri, A., Gage, F., Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells+express+an+immunogenic+nonhuman+sialic+acid.">Human embryonic stem cells express an immunogenic nonhuman sialic acid.</a> Nat Med. 11 (2), 228-232 (2005).</li><li>Kele, M., 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+iPS+cell+line+CTL07-II+from+human+fibroblasts,+under+defined+and+xeno-free+conditions.">Generation of human iPS cell line CTL07-II from human fibroblasts, under defined and xeno-free conditions.</a> Stem Cell Research. 17 (3), 474-478 (2016).</li><li>Uhlin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+iPS+cell+lines+from+monozygotic+twins+in+defined+and+xeno+free+conditions.">Derivation of human iPS cell lines from monozygotic twins in defined and xeno free conditions.</a> Stem Cell Research. 18, 22-25 (2017).</li><li>. Biosafety in Microbiological and Biomedical Laboratories Available from: <a target="_blank" href="https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf">https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf</a> (2009)</li><li>Itskovitz-Eldor, 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=Differentiation+of+human+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Mol Med. 6 (2), 88-95 (2000).</li><li>Chen, K. G., Mallon, B. S., McKay, R. D., Robey, P. 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M., 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+comparison+of+non-integrating+reprogramming+methods.">A comparison of non-integrating reprogramming methods.</a> Nat Biotech. 33 (1), 58-63 (2015).</li><li>. Ideal Xeno-Free Culture Conditions For Human Fibroblasts That Facilitate Efficient Reprogramming Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/posters/xeno-free-culture-conditions-fibroblasts-poster.pdf">https://tools.thermofisher.com/content/sfs/posters/xeno-free-culture-conditions-fibroblasts-poster.pdf</a> (2016)</li><li>Trokovic, R., Weltner, J., Noisa, P., Raivio, T., Otonkoski, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+negative+effect+of+donor+age+and+time+in+culture+on+the+reprogramming+efficiency+into+induced+pluripotent+stem+cells.">Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells.</a> Stem Cell Res. 15 (1), 254-262 (2015).</li></ol>

Il risultato atteso di questo protocollo è la generazione di successo di diverse linee cellulari hiPS derivate clonali. Importante, il metodo per la manutenzione e l&#39;espansione delle cellule hiPS stabilite qui descritto è affidabile e può essere eseguita con poca esperienza precedente della coltura delle cellule staminali. La passaggio di cellule enzimatiche con ROCKi insieme alla matrice LN-521 è noto per mantenere le cellule come cariotipicamente normali, pluripotenti e facilmente capaci di differenziarsi evit...

Dal momento che la prima derivazione delle linee cellulari hiPS da Takahashi et al. 1 , 2 , le cellule hiPS hanno fornito uno strumento utile per la modellizzazione delle malattie, la scoperta di farmaci e come materiale di origine per la generazione di terapie cellulari in medicina rigenerativa 3 . La coltura di cellule hiPS è da tempo dipendente dalla co-coltura con le cellule di alimentazione di fibroblasti 4 , 5 o su Matrigel 6 e con formulazioni di supporto contenenti serum bovino fetale (FBS). Le varianze batch-to-batch sono una conseguenza comune della natura indefinita di queste condizioni di coltura, con conseguente variazioni imprevedibili, che contribuiscono in modo significativo alla inaffidabilità di questi protocolli 7 . Lo sviluppo di un mezzo definito come Essential 8 (E8) 8 e le matrici di colture cellulari definite ad esempio LN-521 9 , consentonoS per la creazione di protocolli altamente riproducibili e aiuti nella robusta generazione e manutenzione di cellule hiPS omologate 7 , 8 , 9 , 10 . Lo sviluppo di tecniche di riprogrammazione gratuite di integrazione è stato un salto in avanti. Originariamente, la riprogrammazione dipendeva da vettori retrovirali che sono stati integrati in modo casuale nel genoma con effetti disruptivi sull&#39;integrità genomica 11 . I progressi nelle metodologie di riprogrammazione includono lo sviluppo di vettori basati su RNA. I vettori RNA hanno un vantaggio rispetto al metodo di riprogrammazione basata sul DNA, poiché l&#39;integrazione involontaria attraverso la ricombinazione genomica non è possibile 12 . I vettori SeV forniscono un&#39;elevatissima e transitoria di fattori esogeni attraverso RNA a singolo filamento senza una fase DNA- 11 . I vettori di riprogrammazione consegnati dal SeVSono diluiti per tutta l&#39;espansione cellulare e, infine, spurgati dalla cultura fornendo un metodo di riprogrammazione libero. Successivamente, la manutenzione della pluripotenza dipende dall&#39;espressione endogena dei geni pluripotenziali 2 . Poiché pionieristici terapie a base di cellule hiPS stanno cominciando a passare a studi clinici, le richieste di lotti standardizzati, la riproducibilità e la sicurezza sono questioni essenziali per affrontare 13 . Pertanto, i prodotti di origine animale devono essere evitati. Ad esempio, l&#39;uso di prodotti xenogeneici è stato associato al rischio di contaminazione degli agenti patogeni non umani. Inoltre, le cellule colte in presenza di componenti animali della coltura animale hanno dimostrato di incorporare acidi silici non umani nelle membrane cellulari che minaccia di rendere le cellule derivate immunogeniche 14 . Quindi, la necessità di eliminare i prodotti xenogeneic è necessaria per qualsiasi futura ricerca clinica. Questo protocollo si applica xeCultura priva di libertà e definizione nel mantenimento delle cellule hiPS che muovono le cellule più vicine alla conformità clinica. Questo protocollo descrive un metodo coerente, altamente riproducibile e facile da usare che genera cellule hiPS standardizzate dai fibroblasti. Offre inoltre un sistema di coltura di facile utilizzo per la manutenzione di cellule hiPS stabilite. Questo protocollo è stato utilizzato per ricavare più di 300 linee di cellule hiPS nel centro nazionale svedese di iPS Core a Karolinska Institutet di cui alcune linee sono state precedentemente descritte 15 , 16 .

<table border="0" cellpadding="0" cellspacing="0" width="711">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dispase</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">171105-041</td>
			<td style="width:223px;">Biopsy digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>Biopsy digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td>Fibroblast matrix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Life Technologies</td>
			<td>21980002-032</td>
			<td>Fibroblast medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Invitrogen</td>
			<td>10270106</td>
			<td>Fibroblast medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PenicillinStreptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Essential 8 media</td>
			<td>ThermFisher Scientific</td>
			<td>A1517001</td>
			<td>iPS cell culture media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LN-521</td>
			<td>Biolamina</td>
			<td>LN521-03</td>
			<td>iPS cell culture matrix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE select 1X</td>
			<td>ThermoFisher Scientific</td>
			<td>12563011</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rho-kinase inhibitor Y27632</td>
			<td>Millipore</td>
			<td>SCM075</td>
			<td>Rho-kinase inhibitor (ROCKi)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CytoTune &#8211; iPS 2.0 reprogramming kit</td>
			<td>Life Technologies</td>
			<td>A1377801</td>
			<td>Sendai virus reprogramming vector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm tissue culture dish</td>
			<td>Sarstedt</td>
			<td>83.3900.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60 mm tissue culture dishes</td>
			<td>Sarstedt</td>
			<td>83.3901.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24 well tissue culture plates</td>
			<td>Sarstedt</td>
			<td>83.3922.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T25 tissue culture flasks</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td>430791</td>
			<td>Centrifuge tubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml tube</td>
			<td>Eppendorf</td>
			<td>0030123.301</td>
			<td>1.5 ml tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CoolCell cell LX</td>
			<td>Biocision</td>
			<td>BCS-405</td>
			<td>Freezing container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650-100ml</td>
			<td>Fibroblast freezing</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Cryovials 1.8 ml</td>
			<td>VWR</td>
			<td>479-6847</td>
			<td>Cryovials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PSC Cryopreservation Kit</td>
			<td>Gibco</td>
			<td>A2644601</td>
			<td>PSC CryomediumRevitaCell supplement</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.05%)</td>
			<td>ThermoFisherScientific</td>
			<td>25300054</td>
			<td>Fibroblast dissociation enzyme</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>31331-028</td>
			<td>Digestive enzymes dilutant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>ThermoFIsher Scientific</td>
			<td>14025-050</td>
			<td>Biopsy preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Haemocytometer</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">BRAND, 718920</td>
			<td>Cell counting</td>
		</tr>
	</tbody>
</table>

integration free derivation of human induced pluripotent stem cells using laminin 521 matrix

Le condizioni prive di Xeno e completamente definite sono parametri fondamentali per una generazione robusta e riproducibile di cellule pluripotenti indotte da umano omogeneo (hiPS). La manutenzione delle cellule hiPS sulle cellule alimentatori o alle matrici non definite sono suscettibili a varianze di lotto, alla contaminazione patogena e al rischio di immunogenicità. Utilizzando la matrice umana ricombinante laminina umana 521 (LN-521) in combinazione con formulazioni di media xeno e definite, riduce la variabilità e consente la generazione costante di cellule hiPS. Il virus Sendai virus (SeV) è un sistema basato su RNA non integrato, evitando quindi le preoccupazioni associate al potenziale effetto dirompente sull&#39;integrità genoma che i vettori integranti possono avere. Inoltre, questi vettori hanno dimostrato un&#39;efficienza relativamente elevata nella riprogrammazione dei fibroblasti dermici. Inoltre, la passaggistica cellulare singola enzimatica agevola la manutenzione omogenea delle cellule hiPS senza una notevole esperienza precedente di ce ce ceCultura. Qui descriviamo un protocollo che è stato ampiamente testato e sviluppato con particolare attenzione alla riproducibilità e alla facilità d&#39;uso, fornendo un modo robusto e pratico per generare cellule umane hiPS definite e prive di xeno dai fibroblasti.

Dalla biopsia alle cellule hiPS L&#39;intero processo dalla biopsia alle cellule hiPS stabilite, lontano dal vettore di riprogrammazione e pronto per la caratterizzazione, richiede circa 16 settimane ( Figura 1 ). Una sequenza più dettagliata è specificata nella Figura 2A . Circa 4 settimane sono necessarie per stabilire e ampliare le colture di fibroblasti. Le prime c...

Watch this Scientific Journal Video about Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix at JoVE.com

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

Una robusta derivazione di cellule pluripotenti indotte da humano (hiPS) è stata ottenuta utilizzando riprogrammazione mediata dei fibroblasti dermici da virus Sendai (SeV) non integrante. La manutenzione delle cellule hiPS e l&#39;espansione clonale sono state eseguite usando condizioni di coltura xeno-free e chimicamente definite con matrice umana laminina 521 (LN-521) e Essential E8 (E8) Medium ricombinante.

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) ...

The overall goal of this protocol is to derive homogenous human induced pluripotent stem cells. This method can be used to derive human induced pluripotent stem cells for many purposes including disease modeling, drug screening, or to derive cells for preclinical cell therapy trials. The advantage of this protocol is that it's robust and generates homogenous cells that are maintained in a user-friendly, Xeno-free, and fully chemically defined culture system.Since human iPS cells recapitulate the genetic background of the donor tissue and can potentially be differentiated into any disease-relevant cell type, they provide excellent tools for thesis modeling. The biopsy dissection and colony picking are intricate procedures. Seeing them performed with experienced hands assists in the understanding of the procedures and makes it easier to repeat.Demonstrating the procedure today will be Harriet RonnHolm and Kelly Day, two highly experienced laboratory technicians from the iPS Core Facility at Karolinska Institutet. Skin biopsy samples can be stored in PBS plus 1%penicillin streptomycin for up to 48 hours at four degrees Celsius, but should be processed as fast as possible after being obtained. Transfer the biopsy to a 35 millimeter dish.Submerge the skin biopsy in a 35 millimeter dish with two milliliters of 70%ethanol for 30 seconds. Add one milliliter of sterile Hank's Balanced Salt Solution supplemented with 1%penicillin streptomycin to a new 35 millimeter dish and transfer the biopsy to the new dish. Transfer the biopsy to a four 35 millimeter dish filled with one milliliter of freshly-prepared 0.1%dispase solution.Using sterile surgical scalpels and forceps, cut the skin biopsy into one to two cubic millimeter pieces. Transfer the biopsy pieces with the dispase solution into a 15 milliliter tube and add an additional two milliliters of dispase. Rinse the 35 millimeter dish with one milliliter of dispase solution and transfer the rinsed solution to the 15 milliliter tube.Incubate the biopsy at four degrees Celsius overnight. On the following morning, add four milliliters of 0.1%collagenase I to the tube with the biopsy pieces and incubate at 37 degrees Celsius for four hours. Thirty minutes prior to seeding the cells, coat one well in a six-well tissue culture plate with one milliliter of 0.1%gelatin in DPBS.Incubate at room temperature for 30 minutes. After four hours of incubation with collagenase, centrifuge the digested cells at 300 times g for three minutes at room temperature. Aspirate the supernatant and resuspend the digest in two milliliters of fibroblast medium.Remove the gelatin solution from the previously prepared six-well tissue culture plate. Transfer the cell suspension including any remaining tissue pieces to the six-well plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide in high humidity.Fibroblasts are ready to be passaged to a T25 tissue culture flask when 80%confluent. Fibroblasts at passage two to four are optimal for reprogramming since reprogramming efficiency is generally higher for lower passage fibroblasts. Aspirate the cell culture medium from the flask and wash the cells with one milliliter of DPBS.Aspirate the DPBS and add one milliliter of 0.5%Trypsin EDTA. Incubate at 37 degrees Celsius for five minutes or until cells are detached. Add two milliliters of fibroblast medium, resuspend the cells, and transfer the cell suspension to a 15 milliliter tube.Centrifuge at 300 times g for three minutes at room temperature. Aspirate the supernatant and resuspend the pellet in two milliliters of fibroblast medium. After counting the fibroblasts using a hemacytometer, seed fives times 10 to the fourth cells in one well of a 24-well tissue culture plate.Incubate cells at 37 degrees Celsius overnight. On the following day, because Sendai virus will be handled, continue the procedure in a designated BSL-2 laboratory while using the appropriate personal protective equipment. Aspirate the cell culture medium and add 250 microliters of Sendai virus solution.Incubate the plate at 37 degrees Celsius. One day before picking the colonies, prepare LN-521 coated 24-well tissue culture plates. Pipette 250 microliters of LN-521 solution into one well of a 24-well plate.Seal the plate using parafilm and incubate at four degrees Celsius overnight. On the following day, aspirate the LN-521 solution from the one well of the 24-well plate and add 500 microliters of E8 into the well. The use of a hairnet and procedure mask is recommended while picking colonies under the stereo microscope.Colonies are ready to be picked when they reach a size of greater than one millimeter in diameter, display sharp edges, and are growing in homogenous monolayers. Do not pick colonies with fuzzy borders and large uncompact heterogeneous cell masses. Cut each colony with a scalpel into smaller pieces in a grid-like pattern.Use a 200 microliter pipette to mechanically scrape the cell sheets from the dish and then transfer the sheet to a well of a 24-well tissue culture plate previously coated with LN-521. Colony selection is critical for the success of this protocol. Picking a few additional colonies will ensure attachment in sufficient numbers.Allow the cells to attach without changing the E8 medium for 48 hours. Enzymatically passage the cells when the colonies have reached a size of greater than five millimeters. Aspirate the cell culture medium from the cells and wash with 250 microliters of DPBS.Aspirate the DPBS and add 250 microliters of dissociation reagent. Incubate at 37 degrees Celsius for three minutes. Observe that the cells have started to round up.Detach cells from the plate by rinsing the cells with dissociation reagent. Add 500 microliters of E8 medium to a 15 milliliter tube and transfer the cells and the dissociation reagent to the tube. Centrifuge at 300 times g for three minutes.Aspirate the supernatant from the 15 milliliter tube and resuspend the cell pellet in 500 microliters of E8 medium. Count the cells using a hemacytometer and seed 2.5 to five times 10 to the fourth cells in another previously prepared LN-521 precoated well containing 500 microliters of room temperature E8 medium. Add ROCK inhibitor to a final concentration of 10 micromolar.Incubate the cells at 37 degrees Celsius. Using this protocol, human induced pluripotent stem cell colonies emerge from transduced fibroblasts at about day 12 post-transduction. Colonies were ready to be picked at about three to four weeks post-transduction.Preferred features include flattened compact cell masses, growth as homogenous monolayers, and sharp edges. These bright field images show an early passage homogenous fully reprogrammed human iPS cell line compared to a highly heterogeneous cell line. The loss of the Sendai virus vector was confirmed by the absence of Sendai virus vector-specific markers in human iPS cells at passage 12 in contrast to human iPS cells at passage three.Immunocytochemistry staining of derived cells for pluripotency markers OCT4, SSEA-4, and NANOG yielded a homogeneously positive result. mRNA expression of endogenous pluripotency genes was shown by reverse transcriptase PCR. Cell cultures containing partially reprogrammed cells that did not express pluripotency factors were discarded.The differentiation potential of human iPS cells was assessed by formation of embryoid bodies. Real-time PCR after 21 days of embryoid body differentiation detected mRNA expression of endoderm, mesoderm, and ectoderm-specific markers which confirmed the derived human iPS cells were capable of differentiation to the three germ layers. Once mastered, this technique can be done with about 22 hours of hands-on time during a time period of approximately 16 weeks.The derived human induced pluripotent stem cells can be differentiated into any cell type of the adult human body to answer any questions relevant to your research field. After watching this video, we hope that you will have a good understanding of how to perform the steps necessary to derive induced pluripotent stem cells from skin biopsies.

Research

JoVE Journal

Developmental Biology

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Derivazione di Cardiomyocytes altamente purificato da cellule staminali umane pluripotenti indotte utilizzando piccoli Differenziazione Molecola-modulato e successiva glucosio Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Ricerca

Biologia dello sviluppo

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

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 ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Quantificazione tridimensionale di spine dendritiche da piramidale neuroni derivate da cellule staminali umane pluripotenti indotte

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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

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 ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Derivazione senza alimentatore di melanociti da cellule staminali umane pluripotenti

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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

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 ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Differenziare i condrociti da cellule staminali pluripotenti indotte dall&#39;uomo derivato dal sangue

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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

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 ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Un modello di Mouse chimerici del fegato umano di ipercolesterolemia familiare facendo uso degli epatociti derivati da cellule staminali pluripotenti indotte

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...