これらの研究は、GJMとGMへのNIH UO1HL107443-01賞によって部分的に資金を供給した。

1。末梢血単核細胞（PBMC）の単離と拡大 0日目<ol><li>クエン酸ナトリウムのBDバキュテイナーCPTの細胞調製チューブに末梢血4mlを描く。チューブ8〜10回転倒し、室温で30分間1800×gで遠心分離します。理想的には、このステップは、採取後2時間以内に行う必要があります。 </li><li>滅菌15mlコニカル遠心管に軟膜（ゲルバリアーとプラズマとの間の細胞層）をピペッティングにより単核細胞（MCS）を収集します。 15分間、300×gで数回、遠心反転、滅菌リン酸緩衝生理食塩水（PBS）で10mlに総量を持参してください。 </li><li>滅菌した10mlのPBSに細胞を再懸濁し、細胞数を実行します。 10分間、300×gで滅菌15mlコニカル遠心管と遠心分離機に1〜2×10 6個の細胞を移す。 </li><li>拡張培地2ml（EM）（50μg/ mlのASCOを含むQBSF-60幹細胞培地で細胞を再懸濁rbic酸、50 ng / mlで、SCF、10 ng / mlのIL-3、2 U / mlのEPOは、40 ng / mlで、IGF-1、1μMのデキサメタゾンおよび100μg/ mlのprimocinまたは1％ペニシリン/ストレプトマイシン）とに転送12ウェルプレートのウェル1。 5％CO 2インキュベーター中、37℃で細胞をインキュベートします。 </li><li> 300×で残った細胞を遠心 10％DMSOを含有FBS中で10分間凍結〜2×10 6細胞/バイアル用グラム。 </li><li>凍結PBMCを使用して、プロトコルを起動するには、10分間、300×gでQBSF媒体および遠心分離機10mlに細胞の1バイアルを解凍します。 EMの2ミリリットル中に細胞を再懸濁し、12ウェルプレートの1ウェルに移す。 5％CO 2インキュベーター中、37℃で細胞をインキュベートします。 </li></ol> 3日目、6日目<ol start="17"><li>滅菌済み15 mlコニカルチューブに細胞を移し、付着細胞を収集するQBSF-60幹細胞培地1mlで一度よく洗ってください。 </li><li> 10分間300 xgで細胞を遠心分離します。 </li><li> EMの2ミリリットル中に細胞を再懸濁し、12の1つのウェルに移すウェルプレート。 5％CO 2インキュベーター中、37℃で細胞をインキュベートします。 </li></ol> 2。 STEMCCAレンチウイルスによる末梢血単核細胞の形質導入 DAY 9 <ol><li>滅菌済み15 mlコニカルチューブに細胞を移し、付着細胞を収集するQBSF-60幹細胞培地1mlで一度よく洗ってください。 </li><li> 10分間300 xgで細胞を遠心分離します。 </li><li> 5ポリブレンとSTEMCCAレンチウイルスのμg/ mlの（MOI = 1から10）、12ウェルプレートのウェル1への転送を含む新鮮なEMの1ml中に細胞を再懸濁する。 （レンチウイルス粒子の産生を記述するプロトコルがで見つけることができます<a href="http://www.bumc.bu.edu/stemcells/protocols/" target="_blank">http://www.bumc.bu.edu/stemcells/protocols/</a> ） </li><li> 90分間25℃で2250 rpmで皿回しをする。 </li><li>スピンした後、新鮮なEMの追加の1ミリリットルを追加 、2mlの培地の合計ポリブレンの5μg/ mlを含有し、37℃でプレートをインキュベート5％CO 2インキュベーター。 </li></ol> 10日目<ol start="16"><li>滅菌済み15 mlコニカルチューブに細胞を移し、付着細胞を収集するQBSF-60幹細胞培地1mlで一度よく洗ってください。 </li><li> 10分間300 xgで細胞を遠心分離します。 </li><li> EMの2ミリリットル中に細胞を再懸濁し、12ウェルプレートの1ウェルに移す。 5％CO 2インキュベーター中、37℃で細胞をインキュベートします。 </li></ol> 3。メッキのMEFに形質導入​​細胞 11日目<ol><li>コート2×10 5細胞で0.1％のゼラチンと板不活性化されたマウス胚性線維芽細胞（MEF）を持つ/ウェルのMEF培地（IMDMを10％FBS、1％非必須アミノ酸を含む、100μMのβ-6ウェルプレートのウェルメルカプトエタノール、2mM L-グルタミン、100μg/ mlのprimocinまたは1％ペニシリン/ストレプトマイシン）。感染当たり3ウェルを準備します。 </li></ol> 12日目<ol start="2"><li>滅菌済み15 mlコニカルチューブに細胞を移し<html>接着した細胞を収集するQBSF-60幹細胞培地1mlで一度よく洗ってください。 </li><li> EM培地（50μg/ mlのアスコルビン酸、50 ng / mlのSCF、10 ng / mlのILで使用したのと同じ濃度で10 ng / mlのbFGFは、アスコルビン酸および成長因子を含むMEF培地3mlに細胞を再懸濁-3、2 U / mlのEPOは、40 ng / mlで、IGF-1、1μMデキサメタゾン）。 </li><li> MEFを含む6ウェルプレートのウェルあたりの細胞のプレート1ミリリットル。 /ウェルの培地を2.5mlの合計のためのbFGF、アスコルビン酸、および増殖因子とMEF培地の1.5 mlを加える。 </li><li> 25℃で500rpmで皿回しをする℃で30分間。 37℃、5％CO 2インキュベーター中で静置します。 </li></ol> 14日目<ol start="6"><li> 10 ng / mlのbFGFおよび50μg/ mlのアスコルビン酸（無成長因子）を含むのMEF培地2.5 mlで一日おきに細​​胞を養う。各フィードで浮遊細胞を吸引して廃棄します。 </li><li> （通常週1回）、必要に応じて追加のMEFを追加します。 </li></ol>P &quot;&gt;〜20日目<ol start="8"><li>かつて小さなコロニーが送り細胞がヒト胚性幹細胞（hESCの）培地（DMEM/F12含む20％ノックアウト血清代替、1％非必須アミノ酸、100μMのβ-メルカプトエタノール、2mMのL-グルタミンの2ミリリットルとの毎日、現れる、100μg/ mlのprimocinまたは1％ペニシリン/ストレプトマイシンおよび10 ng / mlのbFGF）の。 </li></ol> 4。ピッキングとIPSCクローンの拡大 〜DAY 30から40 <ol><li> 12ウェルプレート不活性化されたMEFを含む1ml /ウェルのhESCの培地プラスStemolecule Y27632の1​​0μM（ROCK阻害剤）との事前作成の個々のウェルに各コロニーを拾う。 </li><li>唯一のhESC培地1ml（NO ROCK阻害剤）で、その後毎日の細胞を養う。 </li></ol> 5。多能性マーカーのための免疫蛍光染色<ol><li>染色はミリポアキット &quot;ES細胞マーカーサンプルキット&quot;を使用し、製造業者のプロトコールに従って行った。 </li></ol> 6。 INTEGの切除格STEMCCAベクトル<ol><li>カセットを再プログラミングの切除は、Cre-IRES-Puroの発現ベクターをトランスフェクション後および6に記載されているように簡単なピューロマイシンによる選択を利用して達成されています。 </li></ol>

<ol>
	<li>Lowry, W. E., Richter, L., Yachechko, 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=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Park, I. H., Zhao, R., West, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Takahashi, K., Tanabe, K., Ohnuki, 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=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., 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=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 (2007).</li><li>Stadtfeld, M., Hochedlinger, 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=Induced+pluripotency:+history,+mechanisms,+and+applications.">Induced pluripotency: history, mechanisms, and applications.</a> Genes Dev. 24, 2239-2263 (2010).</li><li>Somers, A., Jean, J. C., Sommer, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transgene-free+lung+disease-specific+human+induced+pluripotent+stem+cells+using+a+single+excisable+lentiviral+stem+cell+cassette.">Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.</a> Stem Cells. 28, 1728-1740 (2010).</li><li>Loh, Y. H., Hartung, O., Li, 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=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., Yuasa, S., Oda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., Dawlaty, M. M., Gao, Q., 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+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Chou, B. K., Mali, P., Huang, X., 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=Efficient+human+iPS+cell+derivation+by+a+non-integrating+plasmid+from+blood+cells+with+unique+epigenetic+and+gene+expression+signatures.">Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.</a> Cell Res. 21, 518-529 (2011).</li><li>vanden Akker, E., Satchwell, T. J., Pellegrin, S., Daniels, G., Toye, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+the+in+vitro+erythroid+expansion+potential+resides+in+CD34(-)+cells,+outweighing+the+contribution+of+CD34(+)+cells+and+significantly+increasing+the+erythroblast+yield+from+peripheral+blood+samples.">The majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.</a> Haematologica. 95, 1594-1598 (2010).</li></ol>

特別な利害関係は宣言されません。

我々は、ここたて​​採取した末梢血数ミリリットルから単離された単核細胞から人間性IPSCを生成するためにSTEMCCAレンチウイルスベクターの使用を記載している。遠い場所から取得されたドナー細胞を利用する際のプロトコルはまた、重要な実用的な含意の詳細（バフィーコートから直接取得した）凍結PBMCを再プログラムするために使用することができます。プログラミングの誘導前に、?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>クエン酸ナトリウムのBDバキュテイナーCPTの細胞調製チューブ</td><td> BD Biosciences社</td><td> 362760 </td><td></td></tr><tr><td> QBSF-60幹細胞培地</td><td>生物学的な質</td><td> 160-204-101 </td><td></td></tr><tr><td> IMDM </td><td>インビトロジェン</td><td> 12440 </td><td></td></tr><tr><td> DMEM/F12 </td><td>インビトロジェン</td><td> 11330 </td><td></td></tr><tr><td> FBS </td><td>アトランタバイオ</td><td> S10250 </td><td></td></tr><tr><td>ノックアウト血清代替</td><td>インビトロジェン</td><td> 10828 </td><td></td></tr><tr><td> Primocin </td><td> Invivogen </td><td> ANT-PM-2 </td><td></td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>インビトロジェン</td><td> 15140 </td><td></td></tr><tr><td> L-グルタミン</td><td>インビトロジェン</td><td> 25030 </td><td></td></tr><tr><td>非必須アミノ酸</td><td>インビトロジェン</td><td> 11140 </td><td></td></tr><tr><td> β-メルカプトエタノール</td><td> MPバイオメディカル</td><td> 190242 </td><td></td></tr><tr><td>アスコルビン酸</td><td>シグマ</td><td> A4544 </td><td></td></tr><tr><td> IGF-1の</td><td> R＆Dシステムズ</td><td> 291-G1 </td><td></td></tr><tr><td> IL-3 </td><td> R＆Dシステムズ</td><td> 203-IL </td><td></td></tr><tr><td> SCF </td><td> R＆Dシステムズ</td><td> 255-SC </td><td></td></tr><tr><td> EPO </td><td> R＆Dシステムズ</td><td> 286-EP </td><td></td></tr><tr><td>デキサメタゾン</td><td>シグマ</td><td> D4902 </td><td></td></tr><tr><td>ポリブレン</td><td>シグマ</td><td> H-9268 </td><td></td></tr><tr><td> bFGFの</td><td> R＆Dシステムズ</td><td> 233-FB </td><td></td></tr><tr><td> Stemolecule Y27632 </td><td>ステムジェント社</td><td> 04から0012 </td><td></td></tr><tr><td> ES細胞マーカーサンプルキット</td><td>ミリポア</td><td> SCR002 </td><td></td></tr></tbody></table>

generation of human induced pluripotent stem cells from peripheral blood using the stemcca lentiviral vector

4つの転写因子の異所性発現は、あるOct4、Klf4の、Sox2、およびCMYCを通して、人間の体細胞は、いわゆる人工多能性幹細胞（性IPSC）1-4を生成し 、多能性状態に変換することができます。患者固有性IPSCは、胚性幹細胞（ESC）を囲む、可能な免疫拒絶を回避したい倫理的な問題を欠いている。したがって、性IPSCは疾患モデル研究、薬理学的化合物のスクリーニング、及び再生治療5のためのかなりの注目を集めている。 私たちは、山中因子6をコードする単一のポリシストロン切除可能レンチ幹細胞カセット（STEMCCA）を使用して異なる肺疾患の患者から導入遺伝子を含まない人間性IPSCの発生を示している。これらIPSC行は皮膚線維芽細胞、再プログラムのために使用される最も一般的な細胞型から生成された。通常は、線維芽細胞を得ることがパンチ生検、細胞の膨張が続く肌を必要とするいくつかの通路のための文化の中での。重要なのは、グループの数は性IPSC 7月9日にヒト末梢血細胞の再プログラム化を報告している。ある研究では、STEMCCAベクトルのTet誘導性バージョンが血液細胞を同時にリバーステトラサイクリントランスアクチベーターをコードして恒常的に活性なレンチウイルスに感染していることが必要とされ、9を採用した。線維芽細胞とは対照的に、末梢血細胞が大幅に患者の不快感や苦痛を減らし、低侵襲の手順を介して収集することができます。構成単切除可能ベクターを用いて血液細胞を再プログラミングするためのシンプルで効果的なプロトコルは、より広範な研究コミュニティがアクセスすることによって、IPSC技術の応用を促進することがあります。さらに、末梢血細胞の再プログラミングすることは皮膚生検を回避すべきで個人からの性IPSCの生成ができるようになります（ すなわち 。瘢痕異常）または起因する既存の疾患状態preventiにパンチ生検にngのアクセス。 ここでは、構成的に4因子を発現する単一フロックス化、切除可能レンチウイルスベクターを用いた末梢血単核細胞（PBMC）から人間性IPSCの世代のためのプロトコルを示しています。 STEMCCAレンチウイルスで形質導入される前を含む培地アスコルビン酸、SCFは、IGF-1、IL-3およびEPOの10,11に記載されているように採取直後または解凍PBMCは9日間に展開されます。次いで、細胞をMEFの上にプレーティングとESCのようなコロニーが2週間後に感染可視化することができる。最後に、選択されたクローンを増殖させ、多能性マーカーSSEA-4、TRA-1-60、TRA-1-81の発現のためにテストされています。このプロトコルは、血液の容易にアクセス可能な4ミリリットルから人間性IPSCの生成のための信頼性のある方法論を提供し、シンプル、堅牢で非常に一貫しています。

我々は、単一のレンチウイルスベクターを用いた末梢血単核細胞から人間性IPSCの生成のためのシンプルで効果的なプロトコルを示しています。 図1Aは、プロトコルの略図を示す。血液はクエン酸ナトリウムとBDバキュテイナーCPTの細胞調製チューブに採取し、遠心分離後、単核細胞はポリエステルゲルと血漿（バフィーコート）（ 図1B）との間のインターフェースか...

Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

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

ここでは、単一のレンチウイルスベクターを用いたプログラミング末梢血の3-4 mlから人間性IPSCの生成のためのシンプルで効果的なプロトコルを示しています。容易に入手可能な血液細胞の再プログラミングすることは、より広い研究コミュニティがアクセスすることによって、IPSCの技術の利用を促進することを約束します。

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, ...

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JoVE Journal

Biology

26.4K ビュー.  Boston University School of Medicine. ここでは、単一のレンチウイルスベクターを用いたプログラミング末梢血の3-4 mlから人間性IPSCの生成のためのシンプルで効果的なプロトコルを示しています。容易に入手可能な血液細胞の再プログラミングすることは、より広い研究コミュニティがアクセスすることによって、IPSCの技術の利用を促進することを約束します。

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

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 ...

生物学

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

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

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 ...

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 ...

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 ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.