We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar&#8217;s Award to JAB.

注：この方法は、内素晴らしいら 10の報告された研究に使用した。 STEMCCA 1.リプログラミング成人ヒト皮膚線維芽細胞<ol><li>解凍成人の皮膚、ヒト線維芽細胞（HUFs）、2分間37℃の水浴中で継代4以下、水とバイアルの上部水没しないように注意しながら。 </li><li> 15mlのコニカルバイアルにヒト線維芽細胞を1mlのスラリーを置き、ゆっくりとジメチルスルホキシド（DMSO）のうち希釈するために、滴下様式で、37℃に予め温めておいた標準HUF培地4mlのを配置し再懸濁細胞。 </li><li>遠心分離を室温で10分間、200×gでバイアル。上清を吸引し、及び/ mlの100,000細胞の懸濁液を作成するために、メディアの適切な容積中で細胞を再懸濁した後、6ウェルプレートの1ウェルに2ミリリットルを追加、0.2％ゼラチンで20分間プレコーティングされた。 注：行当たりのセルの数と同じ数とよく妹と条件がFOを播種する必要があります感染の細胞計数/多重度（MOI）の計算をrを。 </li><li>左右すると前後に細胞を均一に分配するプレート側を揺する。加湿インキュベーター中、37℃、5％CO 2で一晩インキュベートする。 </li><li>形質導入の日に、最初のHUFめっき後ウェル（単数または複数）の姉妹から約24時間後に細胞数（s）を得る。 </li><li> 2×10 8 TU、または同等の、レンチウイルス濃縮物（STEMCCA）を行う解凍し、HUF培地で1×10 8 TU / mlに希釈する。 </li><li> 8 / mlのトランスフェクション剤を補充HUF培地中で、10 MOI比で細胞に形質導入​​する。均等に形質導入されたウェルを混合し、加湿インキュベーター中、37℃、5％CO 2で一晩インキュベートする。 </li><li>レンチウイルス形質導入後の約24時間後には、標準のヒト多能性幹細胞（PSC）を培地にHUFメディアを切り替える。 </li><li>日2〜6日、人間のPSC培地で毎日のメディアを変更。 </li><li> 6日目に、プレートの2 0.2％ゼラチンでコーティングされた10cmのプレートHUF媒体12においてCF1マウス由来の1.5×10 6〜1.75のMEF。 </li><li> 7日目に、注意して、均等にすべてのセルをめっきすることによって、1時16分比率で6ウェルプレート及び通路の1つのウェルから7日目に再プログラミング線維芽細胞をリフトオフする100以上のXG、利用室温トリプシンを遠心分離機にしないMEFの前日に播種した新鮮なヒトPSC培地を有する2つの10cmプレート、。 37°Cで時計回りに螺旋運動と場所で細胞凝集物を分散、5％CO 2で一晩インキュベータに加湿。 </li><li>約48時間後以降のMEFと毎日に再プログラムされた細胞を播種した後、新鮮な培地と一緒に過ごした人間PSCメディアを変更します。 </li><li> 0.2％ゼラチンでコーティングした、個々のウェルの中に特定のコロニーの約8~10個片を配置することにより、3〜4週間後、21ゲージ針で形態のように、典型的なESCとの個々のコロニーをピックアップし、24ウェルプレートに出てサブクローニング24ウェルプレートプレシード人間PSC培地中のCF1マウス由来のMEFとED。 注：採取したコロニーの増殖後、コロニーをタンパク質レベルでの胚様体形成及び多能性マーカーの発現解析によって、多能性として特徴づけされなければならない。 </li></ol> 2.ベクトル統合サイト分析とSTEMCCA切除<ol><li> 10に記載されるように、非限定的な線形増幅PCRによってSTEMCCA組込み部位を分析する。 </li><li>古い人間のESC培地を吸引除去することにより、ゲノム、物品税STEMCCAの所望の領域に1の統合を確実にするためにアッセイすることの後。その後、24時間、標準PSCメディアの3ミリリットルで8 / mlのトランスフェクション剤と濃厚アデノのCre-PuroRウイルスの45μLを兼ね備えています。 </li><li> 24時間のインキュベーション期間の後、混合ウイルス上清を吸引し、ヒトPSC培地で細胞を2回洗浄する。メディア毎日のウィットを変更し、5日間、2μg/ mlのピ​​ューロと一緒に新鮮なヒトPSCメディアを配置H新鮮な培地と抗生物質。 </li><li> 5日後、0.2％ゼラチンでコーティングされたウェル上に21ゲージの針を有するコロニーを、残りのサブクローンは、12ウェルプレートにCF1miceから派生したMEFで予めコーティングし、上記で詳述したように膨張する。 注：凍結ストックを得るために十分な拡張した後、無フィーダー条件への変換が必要です。ほとんどの細胞が正常に形質導入​​されず、インキュベーションの5日間にわたって抗生物質に屈するように少なくとも95％の幹細胞コロニー死を期待する。アデノのCre PuroR因子を含まないコロニーのサンプルをreexposing、感染細胞の宿主染色体への0.001から1パーセントの効率で統合しているがない統合が13を発生していない確認する100％の細胞死を確実にするためにピューロする。 </li><li>マトリックスが液体になるまで約2時間（製造業者の推奨）の期間にわたって、氷上でプレ等分基底膜マトリックス1バイアルを解凍し、冷基本培地中で1：30の最終濃度に希釈するアウト。共同ウェルあたり1mlで6ウェルプレート中のウェルの所望の数で。 1時間室温で放置します。 </li><li> （18ゲージの針を使用して、またはガラスもしくはプラスチック製の「先端」）は、クロスハッチ、少なくとも20〜30個のコロニーよく以前のMEFで被覆されたから。プレートからMEFの高揚と伝達を低減するように注意してください。 <ol><li>滅菌プラスチックピペットチップの簡単な使用のために、火に、より複雑な加熱から、多数の異なるアプローチを介してガラスパスツールピペットの引っ張り、及び仕上げクロスハッチングデバイスを生成するか、私たちの場合のように、 21-および/または18-ゲージの針。 </li></ol></li><li>インキュベーション後にコーティングされたウェルから行列を吸引除去する。 </li><li> 200μlのピペッターで古いプレートから穏やか掻きによってコロニーの部分を削除し、慎重にマトリックスでコーティングされたプレートで新鮮な複合メディア1の3ミリリットルに入れる。 37℃、5％CO 2加湿インキュベーター中で一晩インキュベートする。変化媒体48時間継代後に。 </li><li>市販のDNA抽出キットを使用して、80％以上の密集度で、ポスト切り出し、無フィーダー変換されたhiPSCsからゲノムDNAを抽出します。 </li><li> STEMCCAのレンチウイルスの外因性の統合のための特異的なプライマーとの適切な切除のために分析します。gDNAを-hendo-MycSフォワード、5&#39;-acgagcacaagctcacctct-3 &#39;; gDNAを-hWPREリバース、5&#39;-tcagcaaacacagtgcacacc-3 &#39;。 10μmの原液にPCRグレードの水を用いて凍結乾燥したプライマーを再構成する。 </li><li>以下の5段階のプロトコルを用いてPCRを実行し、3分間95℃での初期変性;以下の各35サイクル：20秒間98℃での変性、15秒間62℃でのプライマーアニーリング、及び15秒間72℃での伸長。 3分間の72℃での単一サイクルの最終伸長が続く。 </li><li>アガロース1.5グラムを秤量し、50mlの1×トリス - 酢酸-EDTA緩衝液中に配置することによって、3％アガロースゲルを作る。アガロースまで、電子レンジを溶解し、PでのDNAゲル染色に配置されているローパー希釈、混合し、室温で1時間凝固させ、ゲルカセットに適切な容積を分配する。 </li><li> PCR産物の負荷12μlを3％アガロースゲルにローディング色素を3μlと混合した。 80 Vで30分間ゲルを実行する</li><li>適切な切除を示すバンドがないことを紫外光を可視化する。 </li></ol>前後の切除したhiPSCsから定量的PCRを用いた遺伝子発現差の3定量<ol><li>商業RNA抽出用キットを用いて、ポスト切り出し、無フィーダー変換されたhiPSCsから全RNAを抽出します。 </li><li>逆転写製造業者の説明書に従って市販のキットを用いて全RNAを1μgまで。アンカーオリゴ（dT）18及びランダムヘキサマープライマーを使用してください。 注：PCRプロトコルは4 KBよりもより多かれ少なかれの遺伝子転写物に合わせて調整されていることを確認してください。特異的プライマーは、いずれかの遺伝子STEMCCAもともと統合Iのために作られる必要がありますNtoを。 </li><li> 20μMストック溶液をPCRグレードの水で凍結乾燥されたプライマーを再構成する。 </li><li>フォワード10μMのUPLプローブ、2Xライトサイクラー480プローブマスター、および20μMで構成され、プライマーを逆反応を20μlあたりのサンプルの5 ngのを使用してください。 </li></ol> GMPグレードの条件に4.コンバージョン<ol><li> GMPグレードの条件にフェイルオーバ後に切除hiPSCsを移行するの初日には、合わせて人間のPSCからなるメディアのブレンドを作るメディア1（複合メディア1）、臨床グレード適正製造基準（GMP）に準拠したメディア（複合メディア2 ）80:20の割合で。 </li><li>吸引物古い複合メディア1とを80:20の比率で新しい複合メディア1及び2、予め温めた3mlのを配置オフ。この特定の比率で3日間、毎日メディアを変更します。バック37℃、5％CO 2インキュベーター中に置き細胞。 </li><li> 4日目に、50：50の割合で混合メディア1と2を作る。古い培地とREPLACオフ吸引除去50:50の比率で複合メディア1及び2を予め温めたメディアと電子。この特定の比は3日間のメディアを変更します。バック37℃、5％CO 2インキュベーター中に置き細胞。 </li><li> 7日目に、20：80の比率で組み合わせたメディア1と2を作る。吸引除去古い培地オフと20:80の比率で組み合わせたメディア1及び2に、予め温めたメディアと交換してください。この特定の比は3日間のメディアを変更します。バック37℃、5％CO 2インキュベーター中に置き細胞。 </li><li> 100比率：10日目に、0で混合メディア1と2を作る。吸引除去古い培地オフと0の組み合わせメディア1及び2に、予め温めたメディアと交換してください：100の比率。この特定の比率で毎日メディアを変更します。細胞は、完全に定義異種非含有のメディアに今ある。バック37℃、5％CO 2インキュベーター中に置き細胞。 注：この全体の変換プロセス中に、18ゲージ針を用いてルーチン継代適切コロニーサイズおよび密度を維持するために必要とされる。 P上のプランコロニーサイズ、フルエンシー、および分化のもとにすべての4から5日間、細胞をassaging。 </li><li>メーカーが推奨するように定義される異種非含有基質と6ウェルプレート中でもコート1。 </li><li>機械的に通過コンフルエントも、すでに18ゲージ針で6ウェルプレートから、異種非含有培地条件に変換する。重要なことは、事前条件メディアに継代する前に、1時間1X ROCK阻害剤を用いて細胞に（組み合わせたメディアは2）新しいメディアを配置。 </li><li>セルに転送されていることを新しいプレートから合成基質溶液を吸引する。旧版からの幹細胞塊を含むすべてのメディアを取り外し、新しいプレートに移す。 </li><li> （一度インキュベータ内部に直接どのような方法でタッチされていない理想的なプレート）バック最小限の物理的擾乱による2日間、37℃、5％CO 2インキュベーター中に置き細胞は最大付着を可能にする。 注：細胞は、毎日のメディアの変更が必要になります。継続的passaginG 4日ごとに重要になります。最適なESC様形態（高い核 - 比）を有するコロニーの一部のみが、21ゲージ針を通路使用。これはよく成長し、最終的に均質なコロニーが得られる適切なhiPSCコロニーのための選択を保証します。コロニーのようなESCの導出に全体的な時間は、初期の複合メディアを変更した後15〜20日の間です。 </li><li> 18ゲージの針を使用して、最初のクロスハッチによりコロニーをすべてGMPグレードのポスト切除hiPSCsダウンフリーズ。 200μlのピペッターを使用してオフにすべてのピースを持ち上げて、4℃で5分間200×gで15ミリリットルコニカルバイアルと遠心分離機に細胞を含むすべてのメディアを転送。 </li><li>細胞は遠心分離されていますが、冷凍メディアは冷たい組み合わせたメディア2等量のからなる7.5％の最終DMSO濃度で凍結培地作る。 </li><li>細胞をペレット化した後、古い培地をオフに吸引し、冷凍メディアでペレットを再懸濁ワイズドロップします。すぐにTRバイアルを凍結し、-80℃の冷凍庫に入れてansfer。 </li></ol> 5.キャラクタライズGMPグレードのポストを切除hiPSCs <ol><li>多能性マーカー、変換後の適切な発現を確実にするために、3 QPCR方法論に従うステップで述べたのと同じ手順で、表1に列挙されたプライマーを使用してキー多能性マーカー（SOX2、OCT4およびNANOG）の発現を定量するためにQPCRを使用してステップ3に記載されているものと同じ手順。 </li><li>使用は、以前に公開10のキットに記載されている標準的な条件に従って、非ヒトシアル酸Neu5Gcを（N -glycolylneuraminic酸）を検出するためにフローサイトメトリー。 </li><li>穏やかに1×リン酸緩衝生理食塩水（PBS）を用いて細胞を細胞培養プレートを洗浄する。 </li><li>室温で5分間1×解離試薬を用いてコロニーを解離する。 </li><li>ステップ5.4の5分間のインキュベーション時間の間に設けられ、ブロッキング溶液（準備氷冷したPBSでのエージェントのブロッキング0.5％とすることでキット）。 </li><li>フローサイトメトリー分析のためのチューブの以下のセットレーベル：未染色を。 4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）DAPI。対照抗体1：200;一次抗体1：200。二次抗体を1：200のロバ抗ニワトリのIgG（H + L）。サンプルのMEF。マトリックス上のサンプルのポストを切除細胞;と合成基質上のポスト切除した細胞のサンプル。 </li><li>穏やかに15 mlのコニカルチューブに溶液および転送コンテンツをブロック2 mlを加えることにより、ステップ5.4からの細胞凝集体を取り除く。 4℃で5分間80×gで遠心し。 </li><li>ステップ5.7で述べたようにソリューションや遠心分離機をブロックで細胞を2回洗浄する。 </li><li>静かに各抗体の対応する希釈体積を有するブロッキング溶液100μl中およそ1×10 6細胞を再懸濁する。穏やかが1時間4℃で揺らしながらインキュベートする。 </li><li>ステップ5.8のように洗浄を3回繰り返します。 </li><li>希釈富栄400μlの細胞ペレットを再懸濁1（キットから）ffer：ステップ5.6の下にリストされているように、チューブ2、6、図7、図8のためのDAPIの100。 </li><li> 40μMストレーナーを通してひずみ細胞とは、フローサイトメーターを介して実行。 </li></ol>

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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=Generation+and+characterization+of+transgene-free+human+induced+pluripotent+stem+cells+and+conversion+to+putative+clinical-grade+status.">Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status.</a> Stem Cell Res Ther. 4, (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receives+FDA+Clearance+to+Begin+World's+First+Human+Clinical+Trial+of+Embryonic+Stem+Cell-Based+Therapy.">Receives FDA Clearance to Begin World's First Human Clinical Trial of Embryonic Stem Cell-Based Therapy.</a> Geron Corporation Press Release. , (2009).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. 64, 3810-383791 (2012).</li><li>Mitani, K., Kubo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+as+an+integrating+vector.">Adenovirus as an integrating vector.</a> Curr Gene Ther. 2, 135-144 (2002).</li><li>Patterson, 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=Defining+the+nature+of+human+pluripotent+stem+cell+progeny.">Defining the nature of human pluripotent stem cell progeny.</a> Cell Res. 22, 178-193 (2012).</li><li>Schroder, A. R., 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=HIV-1+integration+in+the+human+genome+favors+active+genes+and+local+hotspots.">HIV-1 integration in the human genome favors active genes and local hotspots.</a> Cell. 110, 521-529 (2002).</li><li>Hockemeyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+human+pluripotent+cells+using+TALE+nucleases.">Genetic engineering of human pluripotent cells using TALE nucleases.</a> Nat Biotechnol. 29, 731-734 (2011).</li></ol>

James A. Byrne (JAB) and Agustin Vega-Crespo receive research funding from Fibrocell Science, Inc. JAB is a scientific consultant for Fibrocell Science, Inc. No other authors have any competing interests to disclose.

私たちは、要因のないhiPSCsを導出し、将来ヒトの治療における下流細胞分化のためのGMPグレードの条件にこれらの細胞を変換することにより、それらが臨床的に関連することの方法論について説明します。このプロトコルは、種々の細胞型に広く適用可能であるが、我々が原因の患者からの抽出の容易さとパーソナライズされたヒトの治療への適用のために、ヒト皮膚線維芽細胞を再プログ?...

Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions. 
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance. 
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11. 
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:135px;">Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:521px;">Media and reagents</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">DMEM/F12 (basal media)</td>
			<td style="width:111px;">Invitrogen (Carlsbad, CA, USA)</td>
			<td style="width:136px;">11330057</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Fetal bovine serum</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">16000044</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:140px;">Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">11140050</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Glutamax, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">PenStrep, penicillin-streptomycin, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Knockout serum replacement</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">10828028</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Trypsin/EDTA, 0.5%</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15400-054</td>
			<td style="width:135px;">Dilute stock out to 0.05% in 1x PBS</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Basic fibroblast growth factor</td>
			<td style="width:111px;">GlobalStem (Rockville, MD, USA)</td>
			<td style="width:136px;">GSR-2001</td>
			<td style="width:135px;">Reconstitute to 10 &#181;g/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at &#8722;80 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">&#946;-mercaptoethanol</td>
			<td style="width:111px;">Millipore (Billerica, MA, USA)</td>
			<td style="width:136px;">ES-007-E</td>
			<td></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Matrigel (basement membrane matrix)</td>
			<td style="width:111px;">BD Biosciences (San Jose, CA, USA)</td>
			<td style="width:136px;">356231</td>
			<td style="width:135px;">Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">CELLstart (Synthetic Substrate)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1014201</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Stemmolecule Y27632</td>
			<td style="width:111px;">Stemgent (Cambridge, MA, USA)</td>
			<td style="width:136px;">04-0012-02</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Puromycin</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1113802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">LightCycler 480 Probes Master</td>
			<td style="width:111px;">Roche (Basel, Switzerland)</td>
			<td style="width:136px;">4707494001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">ProFreeze-CDM Medium/freezing medium</td>
			<td style="width:111px;">Lonza (Basel, Switzerland)</td>
			<td style="width:136px;">12-769E</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Dimethyl sulfoxide</td>
			<td style="width:111px;">Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td style="width:136px;">D8418</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">PBS</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">14190-250</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">100 BP DNA Ladder</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15628019</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">SYBR Safe DNA Gel Stain 10000x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">S33102</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:140px;">Agarose</td>
			<td style="width:111px;">Bio-Rad Laboratories, Inc. (Hercules, CA, USA)</td>
			<td style="width:136px;">161-3101</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Gelatin, from porcine skin</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">G1890-100G</td>
			<td style="width:135px;">Make stock at 0.2% in PBS, autoclave and store at room temperature</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">mTeSR1</td>
			<td style="width:111px;">StemCell Technologies (Vancouver, BC, Canada)</td>
			<td style="width:136px;">5850</td>
			<td style="width:135px;">Combine Supplement 5X with the basic medium, aliquot and store at 4 &#176;C for up to 2 weeks.&#160;</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Stemedia NutriStem XF/FF Culture Medium</td>
			<td style="width:111px;">Stemgent</td>
			<td style="width:136px;">05-100-1A</td>
			<td style="width:135px;">Thaw at 4 &#176;C O/N, aliquot and store at 4 &#176;C for up to 2 weeks.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Primocin</td>
			<td style="width:111px;">InvivoGen (San Diego, CA, USA)</td>
			<td style="width:136px;">ant-pm-1</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Accutase (Dissociation Reagent)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1110501</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Donkey anti-Chicken IgG AlexaFluor 488</td>
			<td style="width:111px;">Jackson ImmunoResearch (West Grove, PA, USA)</td>
			<td style="width:136px;">703-546-155</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Polybrene/transfection agent</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:136px;">TR-1003-G</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Plasticware</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">12-well plates</td>
			<td style="width:111px;">VWR (West Chester, PA, USA)</td>
			<td style="width:136px;">29442-038</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">6-well plates</td>
			<td style="width:111px;">VWR</td>
			<td style="width:136px;">29442-042</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">10-cm plates</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z688819</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">18-gauge needle</td>
			<td style="width:111px;">Fisher Scientific (Pittsburgh, PA, USA)</td>
			<td style="width:136px;">148265D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">21-gauge needle</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">14-829-10D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Equipment</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD LSRII Flow Cytometer</td>
			<td style="width:111px;">KSystem by Nikon (Tokyo, Japan)</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD FACSDiva Version 6.1.3 Software</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Kits</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">PureLink Genomic DNA Mini Kit</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">K182000</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">KAPA HiFi Hotstart ReadyMix PCR Kit</td>
			<td style="width:111px;">KAPA Biosystems (Wilmington, MA, USA)</td>
			<td style="width:136px;">KK2601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">High Pure RNA Isolation Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">11828665001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:140px;">Transcriptor First Strand cDNA Synthesis Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">4379012001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Sialix anti-Neu5Gc Basic Pack Kit</td>
			<td style="width:111px;">Sialix (Newton, MA, USA)</td>
			<td style="width:136px;">Basic Pack</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 1</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts mTeSR1 and Nutristem</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 2</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts TeSR2 and Nutristem</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:140px;">HUF Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Dulbecco&#8217;s modified Eagle&#8217;s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:140px;">Human Pluripotent Stem Cell Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x &#946;-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;">DMEM/F12, Dulbecco&#8217;s modified Eagle&#8217;s medium/F12; PBS, phosphate-buffered saline.</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
	</tbody>
</table>

derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions

ヒト誘導多能性幹細胞（hiPSCs）は、レンチウイルスベースの再プログラミング方法論を用いて生成することができる。しかし、ゲノムの積極的に転写領域内に残っている可能性の発癌性遺伝子の痕跡は、人間の治療への応用1で使用するための彼らの可能性を制限する。さらに、治療に関連する誘導体に幹細胞のリプログラミングまたは分化由来の非ヒト抗原は、ヒト臨床状況2において使用されるこれらのhiPSCsを排除する。このビデオでは、外因性の導入遺伝子の自由要因フリーhiPSCsを再プログラムし、分析するための手順を提示する。これらhiPSCsその後、レンチウイルスを含む特定のイントロンにおける遺伝子発現異常を分析することができる。この分析は、以前に3遺伝子発現の差を検出するために使用されるあまり敏感技術に勝る利点を有する敏感な定量的ポリメラーゼ連鎖反応（PCR）を用いて行ってもよい。への完全な変換臨床グレードの適正製造基準（GMP）の条件は、ヒトの臨床関連性を可能にする。私たちのプロトコルは、別の方法論が提供する現在のセーフハーバー基準による非ヒト抗原にすべての免疫原性リスクを排除する必要があり、人間の治療用途-ためのGMPグレードのhiPSCsを導出するための要因の無特徴付けられたhiPSC系誘導体を、拡張し、含まれることを提供しています。このプロトコルは、任意のタイプのレンチウイルス再プログラミングされた細胞に広く適用可能であり、GMPグレードの条件に再プログラミングされた細胞を変換するための再現性のある方法を提供します。

我々はSTEMCCAレンチウイルスベースの再プログラミングアプローチを用いて、臨床グレードの因子フリーhiPSCsを導出するためのプロトコルを提示する。 図1Aは、MEFの層上STEMCCAアプローチを再プログラミングした後に、三つの異なる予め切除hiPSC系統の代表的な写真を示す。 STEMCCAの再プログラミングアプローチの主な利点は、異なる研究グループと場所で、複数の科学者によって?...

Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

導入遺伝子のない人間の人工多能性の導出および特徴は、定義された臨床グレードの条件に細胞株および変換の幹

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

derivation-characterization-transgene-free-human-induced-pluripotent

Research

JoVE Journal

Biology

7.8K ビュー.  University of California, Los Angeles (UCLA). We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. 

ビデオ: Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

生物学

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

ヒト人工多能性幹細胞をトランスフェクトしNucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

大人の線維芽細胞から再プログラムヒト人工多能性幹細胞のコロニーを選択し、分離

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

ヒト心筋細胞の生成：フィーダーフリーヒト人工多能性幹細胞からの分化プロトコル

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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 ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

フローサイトメトリーによる高効率心筋細胞へのヒト多能性幹細胞の分化と特性

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

ヒト胞状卵胞からの系統特異的細胞の抽出、標識、精製

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...