著者は、この研究を支援するため、NIHから次の助成認めることを望む：K08DK082783（AR）、P30DK56465（BTS）、U01HL099993（BTS）、T32HL00715036（SKS）、およびK12HL0806406（SKS）を。とJPマッカーシー財団（AR）。

フレッド·ハッチンソンがん研究センターの治験審査ボードと子どもの &quot;sフィラデルフィアの病院で承認され、インフォームドコンセントを書かれ、末梢血サンプルを採取する前に、患者から得られた。すべての施設のガイドラインが観察された。MEF生成を含む全ての動物実験と奇形腫形成は施設内動物管理使用委員会によって承認された。 PBMCおよび赤芽球の拡大1.フィコール分離 - 0日<ol><li>ダルベッコのリン酸緩衝生理食塩水（DPBS）で1：末梢血1に希釈する。 15ミリリットル丸底ポリスチレンチューブでは、慎重に室温フィコール - ハイパックの3ミリリットルに希釈した血液7mlのレイヤ。 </li><li> 30分間400×gでサンプルを遠心。 </li><li> PBMCをプラズマと曇った白色層として見フィコール - ハイパックとの間のインタフェースに定住しているでしょう。滅菌したトランスファーピペットを使用して、PBを収集1 15ミリリットルコニカルチューブにMCを。 DPBSを用いて10 mlにボリュームを持参してください。 </li><li>血球計を使用して、PBMCをカウント。 </li><li>ピペットで5分間300×gで別々の15ミリリットルコニカルチューブと遠心分離機に2×10 6のPBMC。 90％ウシ胎児血清（FBS）/ 10％ジメチルスルホキシド（DMSO）中で2×10 6のPBMC / mlの濃度で残存する細胞凍結スピンダウンする。 </li><li> QBSF 60無血清培地（光から保護）+ペニシリン/ストレプトマイシン（1％）+アスコルビン酸（50 ng / ml）を+細胞因子（SCF、50 ng / ml）を+インターロイキン3（IL-幹：増殖培地を準備3、10 ng / ml）を+エリスロポエチン（EPO、2 U / ml）を+インスリン様成長因子1（IGF-1、40 ng / ml）を+デキサメタゾン（1μM、光から保護し、新鮮なアリコートを解凍し、各メディア変化する）。 </li><li>作りたての拡張培地2mlおよび再懸濁2×10 6 PBMCを12ウェル組織培養プレートの1ウェルに置き、5の雰囲気を37℃の加湿インキュベーター中でインキュベート％O 2、5％CO 2、90％N 2。 </li><li> 3日目と6日目に、培地を交換してください。 <ol><li>細胞を回収し、残りの細胞を収集するためにQBSF-60の2ミリリットルとよく洗う。 </li><li> 5分間300×gで細胞懸濁液を遠心し、上清を吸引除去する。 </li><li>新しい増殖培地2mlに細胞を再懸濁し、同じ12ウェルプレートに戻す。 </li></ol></li></ol>ヌクレオ2.リプログラミング細胞 - 9日目<ol><li>滅菌した1.5mlのエッペンドルフチューブ（ 表1）に再プログラミングプラスミドをピペット。 </li><li>滅菌1.5ミリリットルエッペンドルフチュー​​ブに補足（メーカーにより供給）とピペットでヌクレオソリューションを混ぜる。 </li><li>ピペット2加湿37℃インキュベーター中で12ウェルプレートの場所の1つのウェルに増殖培地（5％O 2、5％CO 2および90％N 2）の前に細胞を添加する平衡化のための溶液。 </li><li>集める培養した細胞と、残りの細胞を収集するためにQBSF-60の2ミリリットルとよく洗う。 </li><li> 5分間300×gで細胞を遠心し、上清を吸引除去する。 </li><li> DPBS 5mlに再懸濁し、5分間300×gで遠心分離して細胞を洗浄する。 </li><li>上清を吸引除去する。 </li><li>生成気泡を避けるように注意しながら、クレオ液+サプリメント100μlの細胞ペレットを再懸濁する。 </li><li> 、リプログラミングプラスミドを含有するチューブに細胞懸濁液をピペットピペット上下回、およびヌクレオキュベットの底にサンプルをピペット。 </li><li>クレオマシンにキュベットを配置し、プログラムのT-019を実行します。 </li><li>クレオキュベットに（ステップ2.3で平衡化プレートから）予め温め拡大培地100μlを移す。 </li><li>すぐに予め温め増殖培地のウェルにサンプル全体と預金を集める。白、フローティング死んだ細胞の破片を集めることは避けてください。 </li&gt; <li>成長のために加湿した37℃インキュベーター（5％O 2、5％CO 2、90％N 2）にプレートを置き。 </li></ol> 3.メッキフィーダー細胞 - 10日または11 <ol><li> 10日目に、プレートフィーダー細胞。 <ol><li>ゼラチンコート1 6ウェル組織培養プレート。 <ol><li>プレートの各ウェル中にハンクス緩衝生理食塩溶液（HBSS）中の0.1％ゼラチンのピペットから1m </li><li>少なくとも5分間、加湿した37℃のインキュベーターでプレートを置きます。 </li><li>使用直前に、プレートからゼラチン溶液を吸引する。 </li></ol></li><li>予め温めMEF培地（ダルベッコ改変イーグル培地（DMEM）+ FBS（10％）+ペニシリン/ストレプトマイシン（1％））の10ミリリットルに3000 cGyをで照射し、2×10 6マウス胚線維芽細胞の1バイアルを解凍する。 </li><li> 5分間300×gで細胞を遠心し、上清を吸引除去する。 12 MEF培地mlのピペット2mlに細胞を再懸濁Iゼラチンコート6ウェルプレートの各ウェルNtoの。 12日目まで、加湿した37℃インキュベーター（5％O 2、5％CO 2、90％N 2）にプレートを置き。 </li></ol></li></ol> 4.フィーダー細胞上で細胞をプレーティングし、培地を交換 - 12日目<ol><li> 1 15ミリリットルコニカルチューブにヌクレオフェクト細胞を収集し、QBSF-60の2ミリリットルとよく洗浄することにより、残った細胞を収集します。 5分間300×gでサンプルを遠心。 </li><li>準備再プログラミング中：イスコフ改変ダルベッコ培地（IMDM）+ FBS（10％）+非動物L-グルタミン（1mM）を+非必須アミノ酸（NEAA、1％）+ペニシリン/ストレプトマイシン（1％）+β β-メルカプトエタノール（0.1ミリモル）+塩基性線維芽細胞成長因子（bFGF、摂食、10ng / mlの新鮮な追加）+アスコルビン酸（摂食、50μg/ mlの新鮮な追加された）。 </li><li>上清を吸引し、リプログラミング培地12ミリリットルで細胞を懸濁します。細胞懸濁液をウェルあたりピペットを2mlにフィーダー細胞の6ウェルプレート（セクション3で調製した）。室温で30分間、50×gでプレートを遠心する。 </li><li>成長のために加湿した37℃インキュベーター（5％O 2、5％CO 2、90％N 2）にプレートを置き。 IPSCコロニーは（1-2週間）に出現し始めるまで、一日おきにリプログラミングミディアムに変更します。 </li><li>準備したhESC培地：DMEM / F12 1：1 +ノックアウト血清代替（20％）+ペニシリン/ストレプトマイシン（1％）+ NEAA（1％）+非動物L-グルタミン（1mM）を+ピルビン酸ナトリウム（1％） +炭酸水素ナトリウム（0.12％）+βメルカプトエタノール（0.1 mM）の+のbFGF（給餌、10ng / mlの新鮮な追加） </li><li> HDAC阻害剤を準備します（給餌で​​新鮮な追加、100μM）酪酸ナトリウム、suberanilohydroxamic酸（SAHAは、200 nMの、給餌で新鮮な追加された）。 IPSCコロニーが出現したら、毎日培地を交換、hESCの培地+ HDAC阻害剤で細胞を供給し始める。 NOTE：ナトリウム酪酸プラスチックチューブに吸着する小分子であり、tはherefore 4℃でホウケイ酸ガラス管に入れて保管してください。 </li></ol> 5.切り分けIPSCクローン<ol><li> IPSCのコロニー（20-25細胞）を単離するのに十分な大きさになったら、P20ピペットでそれらを選ぶ。 </li><li> HDAC阻害剤の準備：酪酸ナトリウム（給餌、100μMで新鮮な追加された）を、SAHAは（摂食、200 nmで新鮮な追加） </li><li>のhESC培地+ HDAC阻害剤+クローニングとリカバリサプリメントを含む個々の35mm皿でiMEFs上に分離したコロニーを置きます。 </li><li>コロニーをピッキングした翌日には、ヒトES細胞培地+ HDAC阻害剤に培地を変更します。 </li><li> IPSCコロニーは継代のために準備が整うまで、毎日培地を変更してください。 </li><li>コロニーが継代2-3で安定いったん媒体からHDAC阻害剤を除去します。 </li></ol>

<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>Koche, R. 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=Reprogramming+factor+expression+initiates+widespread+targeted+chromatin+remodeling.">Reprogramming factor expression initiates widespread targeted chromatin remodeling.</a> Cell Stem Cell. 8 (1), 96-105 (2011).</li><li>Baum, C., Kustikova, O., Modlich, U., Li, Z., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+and+oncogenesis+by+chromosomal+insertion+of+gene+transfer+vectors.">Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors.</a> Human Gene Therapy. 17 (3), 253-263 (2006).</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 (7151), 313-317 (2007).</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 (5928), 797-801 (2009).</li><li>Hong, 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=Suppression+of+induced+pluripotent+stem+cell+generation+by+the+p53-p21+pathway.">Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.</a> Nature. 460 (7259), 1132-1135 (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 (5), 409-412 (2011).</li><li>Yates, J. L., Warren, N., 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=Stable+replication+of+plasmids+derived+from+Epstein-Barr+virus+in+various+mammalian+cells.">Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells.</a> Nature. 313 (6005), 812-815 (1985).</li><li>Ware, C. B., 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=Histone+deacetylase+inhibition+elicits+an+evolutionarily+conserved+self-renewal+program+in+embryonic+stem+cells.">Histone deacetylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells.</a> Cell Stem Cell. 4 (4), 359-369 (2009).</li><li>Mali, 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=Butyrate+greatly+enhances+derivation+of+human+induced+pluripotent+stem+cells+by+promoting+epigenetic+remodeling+and+the+expression+of+pluripotency-associated+genes.">Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.</a> Stem Cells. 28 (4), 713-720 (2010).</li><li>Azuara, 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=Chromatin+signatures+of+pluripotent+cell+lines.">Chromatin signatures of pluripotent cell lines.</a> Nature Cell Biology. 8 (5), 532-538 (2006).</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>Yoshida, Y., Takahashi, K., 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=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5, 237-241 (2009).</li><li>Abbondanzo, S. J., Gadi, I., Stewart, C. L. <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+Embryonic+Stem+Cell+Lines.">Derivation of Embryonic Stem Cell Lines.</a> Methods in Enzymology. 225, 803-823 (1993).</li><li>Kim, H. J., Bae, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors:+molecular+mechanisms+of+action+and+clinical+trials+as+anti-cancer+drugs.">Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs.</a> American Journal of Translational Research. 3 (2), 166-179 (2011).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言。

このプロトコルを使用するときに成功したIPSCの生成のために、考慮すべきいくつかの重要な注意事項があります。偏差が目標前駆細胞集団の非効率的な刺激およびIPSCの生成の低い効率につながる可能性がある赤芽球の膨張段階で、培地交換スケジュールは厳密に従わなければならない。各培地交換新鮮デキサメタゾンで新しい増殖培地を作ることが重要である。そしてQBSF-60基本培地および?...

iPS細胞は、多能性遺伝子の最小限のセットの異所性発現を介して細胞組織から誘導される。この技術は、最初に非常に多能性状態1で表現されるOCT4、SOX2、KLF4、およびcMYCはヒト線維芽細胞のレトロウイルス形質導入によって実証された。これらの一時的に発現「再プログラミング因子」は、ヒト胚性幹細胞2に、標的細胞のエピジェネティックな風景や遺伝子発現プロファイルが類似して変更する。作成後は、性IPSCは、潜在的に、さらなる調査のための任意の組織型に分化させることができる。したがって、それらは、再生医療、疾患モデル、および遺伝子治療用途において使用するための有望。しかし、統合ウイルスのゲノムを破壊することは、内因性遺伝子発現、細胞の表現型の影響を変化させる可能性があり、最終的に科学的な結果にバイアスをかける。さらに、ランダムウイルス統合は有害な携帯電子につながる可能性悪性形質転換3または発癌導入遺伝子4の再発現の可能含めffects、。将来の臨床応用は、非組み込みIPSCの生成が必要になります。 染色体外環状DNA分子であるエピソーム、効果的なコストを発生させるための戦略を提供し、統合のないiPS細胞5。 表1に示したエピソームベクターの組み合わせは、再プログラミングは、OCT4、SOX2、KLF4、MYCL、およびLIN28A因子発現する。 pCXLE_hOCT3 / 4-shp53-Fプラスミドは、細胞リプログラミング6を強化するため、TP53の一時的な抑制のためのp53 shRNAを含まれています。複製欠損pCXWB-EBNA1ベクターは、EBNA1式 7の一過性の増加を提供することで、初期化因子の増幅および増加初期化の効率を促進する。 pCXLE_EGFPプラスミドはDETEの目的のためにヌクレオ混合物に加えることができるトランスフェクション効率をrminingまたはセルソーティングアプリケーションに最適です。 p個のCXWB-EBNA1を除いて、このプロトコルで使用されるエピソームプラスミドは、宿主細胞の分裂8中エピソームの複製およびパーティションを媒介する、ウイルス複製およびEBNA1遺伝子のエプスタイン-バーウイルス起点を含む。エピソームは自然に連続したIPSC拡張7で失われます。サブクローニングおよびeGFP発現の損失から推測できるエピソームベクターの損失を有するiPS細胞の特徴付けは、将来の臨床応用のための完全に統合フリー性IPSCをもたらすことができる。 IPSCの生成のプロセスに固有の系統特異的遺伝子および多能性関連遺伝子の再活性化の抑制である。遺伝子発現の調節は、転写因子、調節DNAエレメント、およびRNAポリメラーゼのアクセスが対象とすることを可能にするDNAとクロマチンの修飾を含む、核内の複数のレベルで生じる遺伝子。グローバルクロマチン修飾を介したエピジェネティックな風景のリモデリングは、多能性遺伝的プログラムの再発現に重要なコンポーネントです。遺伝子発現の調節に重要である特定のクロマチン修飾は、ヒストンDNAコイルの減少緊張を通して遺伝子を標的とするためのアクセスを許可する特定のリジン残基でのヒストンのアセチル化である。 HDAC阻害剤は、アセチル化状態11を支持する可能性が、IPSCの再プログラミングおよびhESCの自己再生9,10を増強することが示されている小分子である。統合レンチ12を用いて、公開前から適合以下に記載されるプロトコルは、エピソームおよびHDAC阻害剤を用いて、末梢血から最適化IPSCを生成するためのステップバイステップの方法を提供する。ここで使用されるHDAC阻害剤の濃度は、ウェアによって記載されたものの半分であるら 9、日常標準にわたって完全に再プログラムIPSCコロニーにおける2倍の増加につながっているHDAC阻害剤なしでエピソーム再プログラミングプロトコル。再プログラミングのこのレベルは、我々は、レンチウイルスの方法で観察する効率と同等である。このプロトコルを用いて、効率的に年齢87歳と同じくらい古い個人からのIPSCを生成した。

<table border="0" cellpadding="0" cellspacing="0" width="1100">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name of Reagent/ Equipment</td>
			<td style="width:151px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:503px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ficoll-Paque PLUS</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">45-001-750</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DPBS</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">RPMI 1640</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10437028</td>
			<td>Store at -20&#176;C until needed. Thaw, aliquot, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dimethyl sulfoxide (DMSO)</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">154938</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">QBSF-60 serum-free medium</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">50-983-234</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">penicillin/streptomycin</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">15140122</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell Line Nucleofector Kit V</td>
			<td style="width:151px;">Lonza</td>
			<td style="width:119px;">VCA-1003</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2% gelatin solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1393</td>
			<td>Store at 4&#176;C, liquify in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Hank&#39;s Balanced Saline Solution (HBSS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14175-103</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:329px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">12440-061</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">L-glutamine, 200 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">non-essential amino acids (NEAA), 100X</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td>Store at 4&#176;C, away from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM/Ham&#39;s F12, 1:1</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">SH30023.02</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">KnockOut Serum Replacement</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium pyruvate, 100 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11360-070</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium bicarbonate, 7.5%</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25080094</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">basic fibroblast growth factor (bFGF)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">PHG0263</td>
			<td>Make 10 mg/mL stock in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium butyrate&#160;</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887-1G</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">hES Cell Cloning &#38; Recovery Supplement</td>
			<td style="width:151px;">Stemgent</td>
			<td style="width:119px;">01-0014-500</td>
			<td>Store at -20&#176;C until needed. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ESC-qualified BD matrigel</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">35-4277</td>
			<td>Thaw overnight on ice at 4&#176;C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20&#176;C until needed. Thaw at 4&#176;C, use immediately.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">StemSpan SFEM&#160;</td>
			<td style="width:151px;">STEMCELL Technologies</td>
			<td style="width:119px;">9650</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ascorbic acid, powdered</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4403-100MG</td>
			<td>Make 5 mg/mL stock in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human stem cell factor (SCF)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">255-SC-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human interleukin 3 (IL-3)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">203-IL-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">erythropoietin (EPO)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">287-TC-500</td>
			<td>Make 1000 U/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">recombinant human insulin-like growth factor 1 (IGF-1)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">291-G1-200</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">&#946;-mercaptoethanol</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td>Make 1000X stock (100 mM) in DPBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dexamethasone</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4902-25MG</td>
			<td>Make 50X stock (50 &#956;M) in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">SAHA (vorinostat)</td>
			<td style="width:151px;">Cayman Chemical</td>
			<td style="width:119px;">149647-78-9</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">12 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">15 mL conical tube</td>
			<td style="width:151px;">Sarstedt</td>
			<td style="width:119px;">62553002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">1.5 mL Eppendorf tube</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">35 mm tisue culture plates</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">10 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">5 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">20 &#956;L pipette tips, barrier tips</td>
			<td style="width:151px;">Genessee</td>
			<td style="width:119px;">24-404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">glass Pasteur pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pipette aid</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-681-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hOCT3/4-shp53-F</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hSK</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hUL</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-EGFP</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXWB-EBNA1</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">37624</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient ips cell generation from blood using episomes and hdac inhibitors

この原稿を効率的にエピソームプラスミドおよびヒストンデアセチラーゼ（HDAC）阻害剤を用いて末梢血からの統合を含まないヒト誘導多能性幹細胞（iPS細胞）を作成するためのプロトコルを示す。このアプローチの利点は、（1）原料として、末梢血の最小量の使用。 （2）リプログラミングベクター非統合。 （3）ベクトルフリー性IPSCを生成するための費用効果的な方法。 （4）シングルトランスフェクション。 （5）小分子の使用は、エピジェネティックな再プログラミングを容易にする。簡単に述べると、末梢血単核細胞（PBMC）ルーチン瀉血サンプルから単離し、次いで、再プログラミングに著しく適している、高度に増殖性の赤血球前駆細胞集団を得た定義された成長因子の中で培養される。非統合、OCT4、SOX2、KLF4、MYCL、LIN28A、およびp53の短いヘアピン（SH）RNAを発現する非伝播エピソームのプラスミドはintroduですシングルクレオによって派生赤芽球へのced。強化された緑色蛍光タンパク質（EGFP）を発現するエピソームの同時トランスフェクションは、トランスフェクションされた細胞の容易な同定を可能にする。エプスタイン-バー核抗原1（EBNA1）を発現する独立した複製欠損プラスミドはまた、エピソームのタンパク質の増大した発現のための反応混合物に添加される。トランスフェクションされた細胞は、その後の継続的再プログラミングのために照射したマウス胚性線維芽細胞（iMEFs）の層上にメッキされている。否やIPSC様コロニーはヌクレオ後約12日目に現れるように、HDAC阻害剤は、エピジェネティックなリモデリングを促進するために培地に添加される。我々は、HDAC阻害剤を含めることが日常的に2倍にすることによって完全に再プログラムIPSCコロニーの生成を増加させることを見出した。 IPSCのコロニーは典型的なヒト胚性幹細胞（hESCの）形態を示したら、それらを穏やかに継続的な成長および増殖のための個々のiMEFコーティングした組織培養プレートに移す。

三日後、およびヌクレオiMEFsにヌクレオフェクト細胞を播種する前に、成功したヌクレオの効率は、eGFPのために蛍光顕微鏡によって推定されるべきである。 図1のeGFPを発現する全細胞集団の約5〜10％の典型的なヌクレオ実験を示す。 再プログラムIPSCコロニーは約2週間後にヌクレオ表示されるようになります。コロニーが明確に定義された境界線を有するほ?...

Watch this Scientific Journal Video about Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors at JoVE.com

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

ここでは、再プログラミング戦略とヒス​​トンデアセチラーゼ阻害剤ベースのエピソームを用いて末梢血からのヒト人工多能性幹細胞を生成するためのプロトコルを記述している。

エピソームおよびHDAC阻害剤を用いて血液からの効率的なiPS細胞の生成

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

efficient-ips-cell-generation-from-blood-using-episomes-hdac

Research

JoVE Journal

Biology

12.7K ビュー.  Fred Hutchinson Cancer Research Center. ここでは、再プログラミング戦略とヒス​​トンデアセチラーゼ阻害剤ベースのエピソームを用いて末梢血からのヒト人工多能性幹細胞を生成するためのプロトコルを記述している。 

ビデオ: Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

アメリカカブトガニ、カブトガニポリフェムスから採血

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

生物学

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

マウス神経組織から単細胞懸濁液の生成

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

STEMCCAレンチウイルスベクターを用いた末梢血からのヒト人工多能性幹細胞の生成

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Multititreプレートフォーマットでヒト多能性幹細胞から神経細胞の迅速かつ効率的な生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

筋ジストロフィーのマウスモデルから分離された骨格筋の等尺性と偏心力発生アセスメント

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

高解像度メルト解析を用いたPCRを使用して、迅速かつ効率的なゼブラフィッシュ遺伝子型決定

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

ヒト骨格筋からの血管由来の多能前駆体の単離

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with 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 include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

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 ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

ヒトiPS細胞のマイクロRNA発現プロファイル、IPから派生網膜色素上皮、および胎児の網膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...