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

この作業は、SSF（B13-0074）とVINNOVA（2016-04134）の資金提供機関によってサポートされました。

患者物質の収集およびhiPS細胞の誘導は、2012年3月28日のストックホルム倫理審査委員会の登録番号：2012 / 208-31 / 3によって承認されています。細胞培養ステップは、特に明記しない限り、バイオセーフティキャビネットで行う必要があります。細胞を扱うときは、常に無菌操作技術を実践してください。開始前に培地、プレートおよび試薬を室温に戻す。高湿度下、37℃、5％CO 2で細胞...

<ol>
	<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>Jin, Z. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pluripotent+stem+cell+culture:+considerations+for+maintenance,+expansion,+and+therapeutics.">Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.</a> Cell Stem Cell. 14 (1), 13-26 (2014).</li><li>Jacobs, 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=Higher-Density+Culture+in+Human+Embryonic+Stem+Cells+Results+in+DNA+Damage+and+Genome+Instability.">Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability.</a> Stem Cell Reports. 6 (3), 330-341 (2016).</li><li>Schlaeger, T. 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>

このプロトコールの予想される結果は、いくつかのクローン的に誘導されたhiPS細胞株の生成が成功したことである。重要なことに、ここに記載された確立されたhiPS細胞の維持および増殖のための方法は信頼性があり、幹細胞培養の経験がほとんどない状態で実施することができる。 ROCKiとLN-521マトリックスとの酵素的単一細胞継代は、細胞を核型正常、多能性および容易に分化させること?...

Takahashi らによるhiPS細胞株の最初の誘導以来、 1、2 、hiPS細胞は、疾患モデル化、創薬、および再生医療における細胞療法を生成するための原材料として有用なツールを提供している3 。 hiPS細胞培養は、線維芽細胞フィーダー細胞4,5またはマトリゲル6および胎児ウシ血清（FBS）を含有する培地製剤との共培養に長く依存してきた。バッチ間の変動は、これらの培養条件の未定義の性質の共通の結果であり、予測不可能な変動をもたらし、これらのプロトコルの信頼性に大きく寄与する7 。 Essential 8（E8） 8および定義された細胞培養マトリックス、例えばLN-521 9のような規定された培地の開発は、高度に再現可能なプロトコールの確立および均質なhiPS細胞の堅牢な生成および維持を助けるためのものである7,8,9,10。 統合型の再プログラミング技術の開発は飛躍的な進歩を遂げています。もともと、再プログラミングは、ゲノムの完全性に破壊的な影響を与えてゲノムにランダムに組み込まれたレトロウイルスベクターに依存していました11 。再プログラミング方法の進歩には、RNAに基づくベクターの開発が含まれる。 RNAベクターは、ゲノム組換えによる意図しない組込みが不可能であるため、DNAベースの再プログラミング方法よりも有利である12 。 SeVベクターは、DNA相11を持たない一本鎖RNAを介して、外因性因子の高い一過性の発現を提供する。 SeVによって送達された再プログラミングベクター細胞の増殖の至るところで希釈され、最終的に培養から脱落して、フットプリントの自由な再プログラミング方法を提供する。その後、多能性の維持は、多能性遺伝子の内在的発現に依存する2 。 先駆的なhiPS細胞ベースの治療法が臨床試験に移行し始めているため、標準化されたバッチ、再現性、および安全性に対する要求は、 13に対処するための重要な課題です。したがって、動物起源の製品は避けるべきである。例えば、異種産物の使用は、非ヒト病原体汚染のリスクと関連している。また、動物由来の培養成分の存在下で培養された細胞は、非ヒトのシリア酸を細胞膜に取り込ませ、誘導された細胞を免疫原性にすると脅かされている14 。したがって、将来の臨床研究には、異種産物を排除する必要があります。このプロトコルはxeを適用しますhiPS細胞の維持における無細胞で規定された培養は、細胞を臨床コンプライアンスに近づける。 このプロトコルは、線維芽細胞から標準化されたhiPS細胞を生成する、一貫性があり、再現性があり、使い易い方法を記載しています。また、確立されたhiPS細胞の維持のための使いやすい培養システムを提供します。このプロトコールは、Karolinska Institutetのスウェーデン国内ヒトiPSコア施設で、300以上のhiPS細胞株を誘導するために使用されています。

<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

Xeno-freeおよび完全に定義された条件は、均一なヒト誘導多能性幹（hiPS）細胞の堅牢で再現性のある世代のための重要なパラメータである。フィーダー細胞または未定義マトリックス上のhiPS細胞の維持は、バッチの変動、病原性の汚染および免疫原性のリスクに影響されやすい。定義された組換えヒトラミニン521（LN-521）マトリックスを異種非含有培地および規定培地と組み合わせて使用​​することにより、変動性が減少し、hiPS細胞の一貫した生成が可能になる。センダイウイルス（SeV）ベクターは、非組み込み型RNAベースのシステムであるため、組み込みベクターが有するゲノムの完全性に対する潜在的な破壊的効果に関連する懸念を回避することができる。さらに、これらのベクターは真皮線維芽細胞の再プログラミングにおいて比較的高い効率を示した。さらに、細胞の酵素的な単細胞継代は、実質的に以前の経験のないhiPS細胞の均質な維持を容易にする培養する。ここでは、再現性と使いやすさに重点を置いて広範囲に試験および開発され、線維芽細胞から定義された異種非含有ヒトhiPS細胞を生成するための堅牢で実用的な方法を提供するプロトコールについて説明します。

生検からhiPS細胞まで生検から確立されたhiPS細胞までの全過程は、再プログラミングベクターがなく、特性決定の準備ができてから約16週間かかる（ 図1 ）。より詳細なタイムラインが図2Aに示されている 。線維芽細胞の培養を確立して拡張するためには、約4週間が必要である。?...

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

ヒトに誘導された多能性幹（hiPS）細胞の強力な誘導は、真皮線維芽細胞の非組込みセンダイウイルス（SeV）ベクター媒介性再プログラミングを使用することによって達成された。 hiPS細胞の維持およびクローン増殖は、組換えヒトラミニン521（LN-521）マトリックスおよびEssential E8（E8）培地を用いた異種非含有および化学的に規定された培養条件を用いて行った。

ラミニン521マトリックスを用いたヒト誘導多能性幹細胞の統合された誘導

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

小分子変調分化とその後のグルコース飢餓を用いたヒト人工多能性幹細胞からの高度に精製された心筋細胞の導出

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

発生生物学

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

髪の由来ケラチノサイトを使用した統合フリーヒト誘導多能性幹細胞の生成

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

ヒト人工多能性幹細胞由来の錐体ニューロンからの樹状突起棘の三次元定量

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

フィーダーフリー条件にセンダイウイルスを用いてヒト末梢T細胞から人工多能性幹細胞の生成

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

ヒト多能性幹細胞からメラノサイトのフィーダーフリーの導出

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

エピソームベクトルを用いたヒト末梢血単核細胞からの統合フリー人工多能性幹細胞の生成

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

末梢血由来ヒト誘導多能性幹細胞からの軟骨細胞の分化

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

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

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

創薬における多能性幹細胞由来肝細胞様細胞を誘導人間を使用して

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

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウス モデル

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