1。再プログラミング<ol><li> 6ウェルプレートのウェル当たり1 × 10 5細胞の密度でシードのBJ細胞。成長培地2mlで培養細胞を（450ミリリットルEMEM 50ミリリットルES修飾FBS、5 mlの10 mMの非必​​須アミノ酸、5 mlのペニシリン/ストレプトマイシン、5ミリリットルに200mM L -グルタミンと0.9ミリリットル55 mMのβを添加した37℃で一晩-メルカプトエタノール）℃、5％CO 2。 </li><li>同じ日に、6ウェルプレートに水で希釈した0.1％ゼラチンを追加し、一晩37℃、5％CO 2をインキュベートすることにより、MEFフィーダープレートの準備を開始。 </li><li>インキュベーション後、BJ細胞から培地を除去し、ポリブレン6μg/ mlのを添加した2 mlの増殖培地を追加し、500μlのhOct4 -レンチウイルス、50μlのhSox2 -レンチウイルス、各ウェルに50μlのhNanog - レンチウイルスと50μlのhLin28 -レンチウイルス。ゆっくりと培地のさえコーティングを確保するためにプレートを揺らし。 37℃および5％CO 2を一晩インキュベートする。 </li><li>同じ日に、液体窒素や融解からCF - 1 MEF細胞の1バイアルを取り外します。ゼラチンコートしたウェルから液体を取り除く（ステップ1.2参照）と0.2 × 10 5細胞/ウェルの密度でMEFフィーダー細胞を加える。ウェルあたりの総体積は（450ミリリットルDMEMに50ミリリットルFBS、5mlの非必須アミノ酸を補充）MEFの増殖培地中の細胞の2ミリリットルにする必要があります。 </li><li>翌日、5分間、200 xgで0.05％トリプシン/ EDTAと遠心機でBJ細胞を切り離します。増殖培地で培地に懸濁するを吸引除去する。 MEFフィーダープレート（ステップ1.4参照）からメディアを削除し、/ウェルBJ細胞懸濁液2 mlを加える。細胞の濃度は約5 × 10 4細胞/ウェルにする必要があります。 37℃および5％CO 2を一晩インキュベートする。 </li><li>ヒトES / iPS細胞の培養液で再播種後培地24時間を取り付けます（400ミリリットルDMEM/F12は100mlのノックアウト血清代替物は、5 mlの10 mMの非必​​須アミノ酸5 mlの200 mMのL -グルタミン、0.9ミリリットルを添加した55 mMのβ-メルカプトエタノールと20 ng / mLのヒト組み換えbFGFを）。 7日間24時間ごとに培養液を交換する。 </li><li> 7日後に、（セクション2を参照）MEF条件培地に培地に変更。 </li></ol> 2。 MEF条件培地の調製<ol><li>シードCF - 1 2 × 10 5細胞におけるMEFフィーダー細胞/ウェルの2ミリリットルMEFの増殖培地中で6ウェルプレートで。 37℃および5％CO 2を一晩インキュベートする。 </li><li>ヒトES / iPS細胞の培養液に培地を変更します。 37℃および5％CO 2を一晩インキュベートする。 </li><li>四日間のために24時間毎に上清を集める。 0.22μmのフィルターを介して上清をフィルターします。 </li><li> bFGFの50 ng / mlの上清を補完する。 </li></ol> 3。 iPS細胞のコロニーの選択と継<ol><li> Stemolecule Y27632の1​​0μMを添加したヒトES / iPS細胞培養培地中でES細胞様コロニーと再シードを選択して細胞培養皿にCF - 1 MEFフィーダー細胞を事前に播種。 37℃および5％CO 2を一晩インキュベートする。 </li><li>最初の7日間の24時間ごとに（Stemolecule Y27632で補足することなく）培地を変更してください。 7日後、（準備のためのセクション2を参照）MEF条件培地に培地に変更。彼らは典型的なヒトES形態を示すまで我々は、細胞を継代続いた。 </li></ol> 4。多能性マーカーの免疫細胞化学的検討<ol><li>細胞をPBSで穏やかに3回洗浄する。 </li><li>室温で20分間、固定液500μlで細胞を固定する。 </li><li>細胞をPBSで穏やかに3回洗浄する。 </li><li>室温で1時間500μlのブロッキングバッファーで非特異的結合をブロックする。 </li><li>一晩4特定の一次抗体を250μlで細胞をインキュベート° C（我々は1:100でTRA - 1 - 81、TRA - 1 - 60、SSEA - 4、SSEA - 3、Oct4の、レトロウイルスベクター、NanogのとLin28を使用希釈）。 </li><li>細胞をPBSで穏やかに3回洗浄する。 </li><li>光から遠ざけることは、室温で1時間二次抗体を250μlで細胞をインキュベートする（我々はヤギ抗マウスIgM Cy3の共役を使用、ヤギ抗マウスIgG Cy3の共役、ヤギ抗ラットIgM抗体Cy3の共役とヤギ抗1:300希釈でウサギCy3の共役）。 </li><li>細胞をPBSで穏やかに3回洗浄する。 </li><li>最後の洗浄のためにDAPIを（最終濃度1μg/ ml）を追加し、核を可視化するために5分間インキュベートする。 </li><li>倍率で細胞を分析する。 </li></ol>パート5：代表的な結果 1。形態の結果： ヒト包皮線維芽細胞（BJ）細胞は、レトロウイルスベクター、Nanogの、そしてLin28 Oct4のと共同で導入した。形態学的変化は、4日後の伝達は早くも観察し、細胞のクラスターは、より緊密に17日目（図1）で満員となった。コロニーは、手動で25日目でピックアップし、CF - 1 MEFフィーダー細胞上で培養した。再プログラミング後のiPS細胞のコロニー形成を容易にするために、我々はStemolecule Y27632を用い各継代時の初期晩播のため、ROCK阻害剤、。良い形態でのiPS細胞コロニーは植民地の3つの連続した​​ラウンド、ピッキングと継代後に観察された。 2。多能性マーカーの発現： さらに分離されたiPS細胞のコロニーを特徴付けるために、我々はES細胞に発現し、共通の多能性マーカーの存在を探した。コロニーは、強いアルカリホスファターゼ（AP）活性（図2）を示した。さらに、免疫細胞化学（ICC）は、表面マーカーTRA - 1 - 81、TRA - 1 - 60、SSEA - 4、SSEA - 3と同様に核のマーカーを含む、多能性マーカー特異的抗体のパネルでiPS細胞のコロニーを行ったOCT4、Sox2、及びNanogの。分離されたiPS細胞のコロニーは、全てのマーカー（図3）陽性であった。 ICCの結果は、iPS細胞は、これらのiPS細胞が密接に未分化ヒトES細胞に似ていることを示している、適切な多能性マーカーの発現パターンを示したことを示している。 <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="図1" /> 図1：形質BJ細胞の形態学的変化。典型的なiPS細胞で形成されたコロニー（）4日間と（B）17日後に形質導入​​の明視野像。 <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="図2" /> 図2：再プログラムBJ細胞のAP活性。三つの異なるコロニーはStemgentアルカリホスファターゼ染色キットで染色。 <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="図3" /> 図3：表面マーカーTRA - 1 - 81、TRA - 1 - 60、SSEA - 4、SSEA - 3、及び核のマーカー10月4日、NanogのとSox2：ヒトiPS細胞は、以下のES細胞特異的マーカーの高いレベルを表現する。

<ol>
	<li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K., 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=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, 861-872 (2007).</li><li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thompson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (1917).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Koyanagi, M., Takahashi, J., Arakawa, Y., Doi, D., Fukuda, H., Hayashi, H., Narumiya, S., Hashimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+Rho/ROCK+pathway+reduces+apoptosis+during+transplantation+of+embryonic+stem+cell-derived+neural+precursors.">Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.</a> J. Neurosci. Res. 86, 270-280 (2008).</li></ol>

この記事の著者は、この記事で使用した試薬と器具を生産するStemgentによって雇用されている。

これらの結果は、ヒト線維芽細胞での転写因子を形質導入StemgentヒトTFのレンチセットが効率的に異所性発現を誘導することにより、iPS細胞のコロニーを生成するために使用できることを示している。プログラミングの実験を設計するとき、いくつかの変数がリプログラミングの効率を最適化するために考慮されるべきである。最初に、アクティブなウイルス対ターゲットの比率（感染MOIの多重度は）最適な導入効率を達成するために主要な情報伝達の段階で変更が必要になることがあります。第二に、標的細胞の成長条件は、リプログラミングに影響する可能性があります。健康と増殖細胞は、リプログラミングに適している。第三に、異なる細胞数のプロトコルを変更する際に、それが標的細胞数が培養皿の表面積に比例して調整することをお勧めします。最後に、このようなY27632として適用するROCK阻害剤は、最近の研究では強化ヒトESコロニーの生存にその有用性が示されたために成功したリプログラミングを確保するために考慮する必要があります。4,5

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<td>Human TF Lentivirus Set</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>00-0005</td>
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<td>Stemolecule Y27632</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>04-0012</td>
<td>&nbsp;</td>
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<td>TRA-1-81 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0012</td>
<td>&nbsp;</td>
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<td>TRA-1-60 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0009</td>
<td>&nbsp;</td>
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<td>SSEA-4 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0003</td>
<td>&nbsp;</td>
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<td>SSEA-3 antibody</td>
<td>&nbsp;</td>
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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

2006年には、山中氏らは最初のOct4のレトロウイルス媒介送達および発現、レトロウイルスベクター、c - MycおよびKLF4はマウスの線維芽細胞の多能性状態を誘導できることを示した。1同じグループはまた、ヒトの体細胞の正常なリプログラミングへの報告レトロウイルスベクターが提供する同じ転写因子の人間のバージョンを使用して人工多能性幹（iPS）細胞が2さらに、ジェームズトムソンらは報告している要因の別のセットのレンチウイルスを介した共発現（Oct4の、レトロウイルスベクター、NanogのとLin28 ）iPS細胞にヒトの体細胞をリプログラミングすることができた3。 iPS細胞は、形態、増殖と身体のすべての組織型に分化する能力のES細胞に類似しています。彼らは胚の破壊の倫理的ジレンマなしでES細胞の主要な特性を示すようにヒトiPS細胞はES細胞上の明確な利点がある。患者固有のiPS細胞の生成は、非自己移植細胞の免疫拒絶反応の可能性を排除することにより、パーソナライズされた再生医療の治療に重要な障害を回避することができます。 ここではStemgentヒトTFのレンチセットを使用して、ヒト線維芽細胞を再プログラミングするためのプロトコルを示す。我々はまた、このセットで再プログラム細胞は形態四日後の伝達のiPSを表示するために始めることを示している。 Stemolecule Y27632を使用して、我々は植民地の3つの連続した​​ラウンド、ピッキングと継代後にiPS細胞と観測された正しい形態のために選択。我々はまた、多能性マーカーAP、表面マーカーTRA - 1 - 81、TRA - 1 - 60、SSEA - 4、およびSSEA - 3、及び核のマーカーOct4の、Sox2、及びNanogのを表示する細胞をリプログラミング後のことを示しています。

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set at JoVE.com

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

我々は、人的要因Oct4は、レトロウイルスベクター、Nanogの、そしてLin28のレンチウイルスを介した配信を用いてヒトの体細胞から誘導多能性幹細胞の生成のためのプロトコルを示す。多分化能は、形態と胚性幹（ES）細胞特異的マーカーの存在によって確認された。

StemgentヒトTFのレンチセットでヒト線維芽細胞を再プログラムすることによって誘導多能性幹細胞の生成

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

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Unit 1

Embryonic and Induced Pluripotent Stem Cells

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29.7K ビュー.  Stemgent. 我々は、人的要因Oct4は、レトロウイルスベクター、Nanogの、そしてLin28のレンチウイルスを介した配信を用いてヒトの体細胞から誘導多能性幹細胞の生成のためのプロトコルを示す。多分化能は、形態と胚性幹（ES）細胞特異的マーカーの存在によって確認された。

ビデオ: Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

生物学

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

4つの転写因子とマウス胚線維芽細胞を再プログラムすることによって誘導多能性幹細胞の生成、ドキシサイクリンは、レンチウイルス伝達系の誘導性

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

ヒト胚性幹細胞を凍結融解

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 ...

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 Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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, ...

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 ...