この作品は、フリードライヒ失調研究アライアンスとアーノルドファミリー財団とMDアンダーソンがんセンターの幹細胞と再生科学総合研究センターからのパイロット助成金によって支えられている。

1。ウイルスの生産と伝達<ol><li> DMEM培地（DMEM高グルコース、不活化10％FBS、熱、2 mM L-グルタミン、抗生物質なし）10ml中〜7-8×10 6％10 cmのプレートの密度でプレートフェニックス周りに細胞。 37一晩インキュベーターや文化の場所°C、5％CO 2。 </li><li>ベクトル符号化OCT3 / 4、Sox2の、KLF4、c-mycの、またはGFP遺伝子（プラスミドAddgene 17217、17218、17219、17220）、35μlのFuGENE 6トランスのいずれかの12μgを用いて次の日にトランスフェクションフェニックス細胞。 DMEM培地（DMEM高グルコース、2 mM L-グルタミン、無FBSおよび抗生物質）500μlのトランスフェクションミックスを調製する。 20分インキュベートします。穏やかに70から80パーセントコンフルエントフェニックス細胞を含む10 cmのプレートにDNA複合体をピペット。 </li><li> 8時間ペニシリンおよびストレプトマイシンを含むDMEM培地（DMEM高グルコース、不活化10％FBS、熱、2 mM L-グルタミン）のトランスフェクション後 - メディア6を交換してください。 </li><li>その後、ウイル​​スを含む培地に4回12時間で収集間隔は、すべての部分を組み合わせて、剥離した細胞と破片を（ウイルス粒子を含むメディアがしかし凍結が推奨されていない場合は、感染活動を失うことなく2週間冷蔵庫に保管することができます）を削除する0.45μmフィルターを用いて濾過した。 </li><li>レトロウイルスのメディアコレクションの最後の日前に凍結ストックの24時間からレトロウイルス形質導入、プレートヒト成人線維芽細胞（流路10、コーリエル医学研究所）のために。シードは、DMEM高グルコースで、ウェルあたり1×10 5細胞の密度で6ウェルゼラチン覆われたプレート上で細胞を、10％FBS、2mMのL-グルタミン、ペニシリン、ストレプトマイシンおよび非必須アミノ酸。 </li><li>フェニックスの細胞から得られたウイルス粒子を含有する培地で翌日代替DMEM培地。のために1600グラムで、線維芽細胞の培養と遠心機の6ウェルプレートの各ウェルにポリブレン6μg/ mlのを補充したウイルスのメディア（それぞれのOct4、Sox2の、KLF4、およびc-mycのメディアを1 ml、4 mlの合計）を追加20℃、1hの約12時間AFTER伝達、線維芽細胞培養培地でウイルスのメディアを交換してください。 24時間間隔でウイルス感染し、遠心分離を3回実行します。 </li><li>最後に、感染後48時間DMEM培地で培養線維芽細胞。 GFPを発現するレトロウイルス媒体と並行して伝達経路による感染の効率を確認してください。図1は、ヒト線維芽細胞の遠心分離促進レトロウイルス感染の効率を示しています。 </li></ol> 2。再プログラミング<ol><li>線維芽細胞増殖培地中で〜2×10 5細胞をウェルあたりの処理ゼラチン6ウェルプレートの密度でMEF細胞を播種することによってγ線照射したMEFフィーダー層6ウェルプレートを準備します。次の日は、線維芽細胞の増殖培地中でウェル当たり1.2×10〜4の密度で0.05％トリプシン/ EDTAとシードそれらを使用して、ヒト線維芽細胞に感染し分割します。 </li><li>次の日は、hES培地（DMEM/F12、20％ノックアウト血清代替物、非必須アミノ酸、penicilliを使用してメディアを交換するN /ストレプトマイシン、2mMのL-グルタミン、0.1mMのβ-メルカプトエタノール、20 ng / mlの基底繊維芽細胞成長因子（bFGF）、リプログラミングの最初の7日間の0.5 mMの酪酸ナトリウムを添加した。毎日のメディアを変更します。 </li><li>毎日の形質細胞の形態学的変化を監視します。 HIPSのような細胞のコロニーは、約5パッケージをemergeすることを開始します - 10日間のフィーダー細胞に形質導入​​した線維芽細胞を転送した後。 hiPSCコロニーは約14を分離する準備ができました - 28日MEFフィーダー細胞上にめっきした後。 </li></ol> 3。 hiPSCコロニーの単離<ol><li> hiPSCコロニーをピッキング前日は、線維芽細胞の増殖培地中でγ線照射したMEFフィーダー細胞（ウェル当たり〜4×10 4細胞）を24ウェルプレートを準備します。この段階でhiPSCコロニーは、マトリゲルとmTeSR1媒体を使用してフィーダーフリーの培養に転送することができます。 </li><li> hiPSCコロニーの分離前には、線維芽細胞成長mediuを削除することにより、MEFプレートを準備mとFBSの痕跡を削除するには、PBSでMEFSをすすぐ。その後、各ウェルに7,8に10μMのROCK阻害剤Y27632で0.5ミリリットルのhES培地（またはマトリゲル被覆したウェルの場合mTeSR1培地）を追加します。 </li><li>必要に応じて、層流フード（ - C図2A）に搭載された顕微鏡下で21ゲージ針でhiPSCコロニーを周囲の線維芽細胞を削除します。 PBSでプレートをすすぎ、TRA-1から81 StainAlive特異抗体（1:200、Stemgent）を含む新鮮なヒトESメディアを追加します。後30分、10μMROCK阻害剤を添加した新鮮なヒトESメディアで抗体を含有する培地を交換してください。蛍光顕微鏡と客観的なマーカー（図2D）を使用してマークTRA-1から81までの正のコロニーの下に板を調べます。 </li><li>図2E及びFに示すように、21ゲージ針を用いていくつかの小さな部分に層流フードで顕微鏡下でTRA-1から81までの正のhiPSCコロニーを、カット</li><li> INDにhiPSCコロニーの自動ピペット（P200）転送の断片を使用して、MEFのまたはマトリゲルを含む24ウェルプレートのウェルにividual。非HIPSセルを転送することは避けてください。 37℃、5％CO 2インキュベーター内でプレートを置き、hiPSCコロニーが24から36時間アタッチすることができます。 </li><li>ヒトESまたはmTeSR1（マトリゲル上に成長したコロニーのための）メディアを毎日変更します。正しいHES-のような形態を有するhiPSCコロニーは24ウェルプレートへの初期転送した後、48時間表示されます。 </li><li> 6ウェルプレート12ウェル、その後、上に8日 - 手動で通過hiPSCコロニー毎に6。 </li><li>クローン性増殖とhiPSCラインを確立した後、図3に示すように、免疫細胞化学を用いた多能性マーカーTRA-1-60、SSEA-4、OCT3 / 4、Sox2、及びNanogの発現を分析します。核型と奇形腫形成の分析6,9を用いて得られた線のゲノムの完全性と分化能を評価します。 </li></ol> 4。代表的な結果レトロウイルス含有培地iの効率的な伝達の成功したリプログラミングの重要な。これは、図1に示すように、効率性を監視するウイルスごとに、単一のプログラミング実験を発現してGFPを用いて全体のトランスフェクション/感染症の手順を行うことをお勧めします。一般的に0.5の範囲であった非濃縮ウイルスメディアを用いたヒト線維芽細胞の情報伝達を介して、10で説明したようにGFP発現ウイルスの力価は、決定- 5×10ミリリットル（VP / ml）のあたり7ウイルス粒子。 線維芽細胞は2日最後の感染後できるだけ早く形態を変更します。トリプシン処理ヒト線維芽細胞は、慎重にMEFフィーダー細胞上に播種する前にカウントする必要があります。これは、各細胞株以来、3つの異なる密度（6×10 3、1.2×10 4、6ウェルプレートの1ウェルあたり2.5×10 4）で種子細胞に推奨されている別の成長特性および播種密度は、プログラミングの効率化のために重要であるを示しています。使用される酪酸ナトリウム最初の7 - プログラミングの14日間hiPSC形成の約5倍の効率を向上させます。頻繁に、感染した線維芽細胞を高い密度で播種した場合は特に、線維芽細胞様細胞は培養皿の生い茂る、図2Aに示すようにhiPSCコロニーをカバーすることができます。このケースでは、線維芽細胞層は慎重に持ち上げてhiPSCコロニー（図2B）を明らかにするために削除することができます。その後、hiPSCコロニーはヒトESメディアでリンスし、TRA-1から81抗体で染色する必要があります。線維芽細胞に応じて、約20 - IPSCのような形態を実証するコロニーの40％は、TRA-1-81抗体で染色されません。 24 - 識別hiPSCコロニーは、次の12内のプレートから転送することができます。長時間のインキュベーションは、hiPSCsの急速な分化になります。コロニーは、手動でMEFフィーダー細胞またはマトリゲルをコートしたプレートのどちらかに継代することができます。展開が確立した後hiPSCクローンpluripotenの発現のための免疫細胞化学（ICC）を使用してテストする必要がありますCYマーカーは、図3に示した。さらに、生成されたiPS細胞の詳細な分子特性を含める必要があります：RT-PCR、9サイレンシングの導入遺伝子の多能性遺伝子および分析のプロモーターのDNAメチル化のデモを使用して多能性遺伝子発現の解析を。 <img alt="図1" src="/files/ftp_upload/3416/3416fig1.jpg" /> 図1。 GFPを発現するレトロウイルスを用いて決定し、ウイルス形質導入の効率が向上します。（A、B）がフリードライヒ失調症患者（GM03665、コーリエル医学リポジトリ）由来のヒト成人線維芽細胞は、GFPレトロウイルスメディアを持つ2つの連続した感染症の後に可視化した。 （C、D）ヒト線維芽細胞は、1600グラムで直接1hの6ウェルプレート上での細胞の遠心分離に続いてGFPレトロウイルス媒体の同じバッチで感染させた。画像は、感染後48時間捕獲された。 <img alt= &quot;図2&quot; SRC = &quot;/ files/ftp_upload/3416/3416fig2.jpg&quot; /&gt; 図2。 hiPSCコロニーの同定および単離（A）線維芽細胞に囲まれたhiPSCsを含むプレートの位相コントラスト画像。細胞はヒトESメディアで21日間培養した。周囲の線維芽細胞層を除去した後（B、C）と同じhiPSCコロニー。 （D）が正しく書き換えhiPSCコロニーはTRA-1から81の表面マーカー抗体を用いたライブ染色により識別され、無菌の注射針（E、F）を使用してカットし、24ウェルプレートの個々のウェルに移した。 <img alt="図3" src="/files/ftp_upload/3416/3416fig3.jpg" /> 図3。多能性特異的なマーカーOCT3 / 4、Nanogの、レトロウイルスベクター、SSEA4とhiPSCsでTRA-1-60の発現は、免疫細胞化学によって決定された。

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特に神経系と神経ヒトの疾患、勉強、特に十分なヒト細胞モデルの到達不能に起因する困難されています。多様な種類の細胞にそれらを区別するために誘導多能性幹細胞および潜在的に容易に得られる体細胞を再プログラムする能力は、遺伝性疾患の細胞モデルを作成する可能性を開いた。さらに、性IPSCは、再生医療の将来非常に有望なものである。したがって、多能性に体細胞を再プログ...

<table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> 11965 </td></tr><tr><td> DMEM/F12 </td><td>インビトロジェン</td><td> 11330 </td></tr><tr><td> KSR </td><td>インビトロジェン</td><td> 10828 </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></tr><tr><td>酪酸ナトリウム</td><td>シグマ</td><td> B5887 </td></tr><tr><td> Y27632 </td><td> Stemgent </td><td> 04から0012 </td></tr><tr><td> bFGFの</td><td> Stemgent </td><td> 03から0002 </td></tr><tr><td> TRA-1-81抗体</td><td> Stemgent </td><td> 09から0069 </td></tr><tr><td> Oct3 / 4の抗体</td><td>サンタクルス</td><td> SC-8628 </td></tr><tr><td> NANOG抗体</td><td>に：Cell Signaling Technology </td><td> 4903S &lt;/ TD&gt; </tr><tr><td> TRA-1-60抗体</td><td>ミリポア</td><td> MAB4360 </td></tr><tr><td> Sox2の抗体</td><td>に：Cell Signaling Technology </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td>ミリポア</td><td> MAB4304 </td></tr><tr><td> CF1のMEF </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td>客観的マーカー</td><td>ニコン</td><td> MBW10010 </td></tr><tr><td>マトリゲル</td><td> BDバイオサイエンス</td><td> 354277 </td></tr><tr><td> mTeSR1 </td><td> StemCellテクノロジーズ</td><td> 05850 </td></tr><tr><td> β-メルカプトエタノール</td><td>シグマ</td><td> M7522 </td></tr><tr><td> FuGENE 6トランスフェ</td><td>ロシュ社</td><td> 11814443001 </td></tr><tr><td>ポリブレン</td><td>シグマ</td><td> H9268 </td></tr><tr><td>オブジェクトマーカー</td><td>ニコン</td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

ここに我々は1-3ナトリウム、酪酸の存在下でOCT3 / 4、Sox2の、KLF4およびc-myc（OSKM）をコードするレトロウイルスベクターを用いたヒト人工多能性幹細胞（hiPSC）にヒト成人線維芽細胞を再プログラミングのプロトコルを提示します。我々は、フリードライヒ失調症患者（GM03665、コーリエル医学リポジトリ）から派生した遅く通路（&gt; P10）ヒト成人線維芽細胞を再プログラムするためにこのメソッドを使用していました。プログラミングのアプローチは、ウイルス含有培地の存在下で線維芽細胞の反復的な遠心分離を用いた高効率伝達プロトコルが含まれています。書き換えhiPSCコロニーがTRA-1-81、多能性細胞の表面マーカーのライブ免疫染色を用いて同定した、非書き換え線維芽細胞から分離し、手動で4,5継代。これらのhiPSCその後、マトリゲルプレートに移し、直接プログラミングプレートから、フィーダーフリーの条件下で増殖させた。最初の通過から始めて、hiPSCのコロニーは、hES-L特性を実証IKE形態。このプロトコルを使用して、選択したコロニーの70％以上が正常に拡大し、細胞株に確立することができます。確立されhiPSC行は表面マーカーTRA-1-60及びSSEA-4と同様に、核のマーカーOCT3 / 4、Sox2、及びNanogの特徴を含む多能性マーカーを表示されます。ここで紹介するプロトコルが確立され、フリードライヒ失調症患者と対照個体6、人間の新生児線維芽細胞と同様に、ヒトケラチノサイトから得られた成人の繊維芽細胞を用いてテストされています。

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

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

我々はとレトロOCT3 / 4、Sox2の、KLF4およびc-myc（OSKM）をコードするベクターとライブ染色により正しく再プログラムhiPSCの識別を用いたヒト人工多能性幹細胞（hiPSC）TRA-にヒト体細胞の効率的なリプログラミングのためのプロトコルを提示1から81抗体。

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 ...

selecting-isolating-colonies-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

19.8K ビュー.  University of Texas M.D. Anderson Cancer Center. 我々はとレトロOCT3 / 4、Sox2の、KLF4およびc-myc（OSKM）をコードするベクターとライブ染色により正しく再プログラムhiPSCの識別を用いたヒト人工多能性幹細胞（hiPSC）TRA-にヒト体細胞の効率的なリプログラミングのためのプロトコルを提示1から81抗体。

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

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 ...

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

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 ...

皮膚生検を使用して患者由来の足細胞を生成する

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

著者スポットライト:皮膚生検からの患者由来足細胞の生成  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

皮膚生検からの患者由来足細胞の生成

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...