我々は、技術支援のために慶應義塾大学医学部から佳子三宅さやかKanaami、Chihana藤田美穂山口、夏子辺見、とレイ大野に感謝します。この研究の一部は健康研究成果の実用化を加速するために、R＆Dシステムズ支援プログラム、および再生医療の実現のためのハイウェイプログラムによって資金を供給されました。

1.活性化されたヒトT細胞を準備します<ol><li> 10ミリリットルを静脈穿刺によってドナーからのヘパリン化全血を採取します。 <ol><li>静脈穿刺の前にドナーのインフォームドコンセントを取得します。静脈穿刺は、資格や訓練を受けた者が行う必要があります。 </li><li>滅菌装置と、次の適切なバイオセーフティ指針に得られた血液サンプルを処理します。 </li></ol></li><li> 10 mlのD-PBSを用いて全血10 mlのヘパリンを希釈（ - ）。 </li><li>新しい50mlのコニカルチューブに15ミリリットルフィコール溶液（フィコール・パックプレミアム）を置きます。 </li><li>ステップ1.3でフィコール液にステップ1.2で作製した20 mlの血液溶液をレイヤ。電動ピペットを用いて、血液溶液は、管の内壁に沿ってゆっくりとステップ1.2の実行を準備しましょう​​。血液溶液とフィコール溶液を混合しないように注意してください。 </li><li>室温で30分間、400×gで遠心分離します。 「遅い」遠心減速度を設定します。 注：遠心分離した後、白い薄いです層は、上部乳白色の血漿層と下部透明なフィコール層との間に現れます。このホワイト薄い層は、単核細胞が含まれています。 </li><li> 1 mlのピ​​ペットを用いて新しい50mlのコニカルチューブに、単核細胞層を転送します。フィコール液を含まないように注意してください。 </li><li> 10ミリリットルの合計体積に単核細胞に、そして室温で5分間、200×gで遠心分離（ - ）D-PBSを追加します。 </li><li>上清を捨てます。単核細胞に、そして室温で5分間、200×gで遠心分離（ - ）のD-PBS 10ミリリットルを追加します。 </li><li> 、上清を捨て、収集した単核細胞にKBM502培地1mlを追加し、血球計数器を用いて細胞数を計測します。 6×10 5以上の単核細胞を、フィコールを用いて適切な分離と全血をヘパリンmlの10から得ることができます。 </li><li> D-PBS中10μg/ mlの濃度の抗ヒトCD3抗体溶液を調製します（ - ）。サーフソーク、6ウェルプレートに、抗ヒトCD3抗体溶液を加えます各ウェルのエース、少なくとも30分間、5％CO 2インキュベーター中、37℃で皿をインキュベートします。 </li><li>各ウェルからの抗ヒトCD3抗体溶液を除去し、D-PBSで1回洗浄（ - ）細胞を播種する直前に。 </li><li> KBM502培地中の抗ヒトCD3抗体でコーティングした6ウェルプレートに2.5×10 5細胞/ cm 2の密度で、ステップ1.9で調製した種単核細胞。 T細胞がコンフルエントに達するまで、培地交換せずに3-7日、5％-CO 2インキュベーター内で37℃でインキュベートします。この期間中のT細胞が両方懸濁し、接着細胞です。 </li></ol>センダイウイルスベクターを用い2.感染ヒトT細胞<ol><li> 3-7日の培養後、ピペッティングすることによって、既存の媒体と活性化T細胞を収集します。室温で5分間、200×gで15 mlのコニカルチューブと遠心分離機にそれらを転送します。 </li><li>上清を除去し、1mlの新鮮なKBM502媒体を追加します。細胞とプレート1をカウントします。5×10 6細胞を、抗CD3抗体でコーティングした6ウェルプレートの各ウェルに（2 mlの新鮮な培地でKBM502）。 </li><li>氷の上に、OCT3 / 4-たSeV /TSΔF、SOX2-たSeV /TSΔF、KLF4-たSeV /TSΔF、およびc-MYC（HNL）-sev /TS15ΔFのセンダイウイルスソリューションを解凍します。 </li><li> -sev /TS15ΔFは、感染の多重度（MOI）の各ウェルに個別にSOX2-たSeV /TSΔF、KLF4-たSeV /TSΔF、およびc-MYC（HNL）、OCT3 / 4のSeV /TSΔFを含むセンダイウイルスソリューションを追加します。さらに24時間、5％-CO 2インキュベーター内で37℃で10インキュベートの。 </li></ol> 3.ヒトT細胞からのSeVベクターを削除します<ol><li> 24時間感染後、ピペットを用いて感染した細胞を収集します。接着細胞が存在する場合、それらは容易に繰り返して上下にピペッティングして取り外すことができます。 15mlのコニカルチューブにそれらを移し、室温で5分間、200×gで遠心します。 </li><li>センダイウイルスベクターを含む上清を除去し、2ミリリットル新鮮なKBM502媒体を追加します。 EACで細胞をReplate井戸の時間。さらに24時間、5％-CO 2インキュベーター中、37℃でインキュベートします。 </li></ol> 4.マトリゲル層上の細胞を再播種<ol><li> 4℃で（この研究で使用される特定のブランドのための材料のリストを参照）マトリックスゲルを解凍し、DMEM / F12培地10mlの0.2 mlで溶解することにより基底膜マトリックス溶液を調製します。 注：マトリックスゲルは、（本研究で用いた特定のブランドのための材料リストを参照してください）​​完全に解凍されるように、4℃でO / Nを解凍する必要があります。使用直前まで氷上で保管してください。 </li><li> 100-mmディッシュにディッシュ当たりプレート5 mlの基底膜マトリックス溶液と、細胞を播種する前に、少なくとも30分間室温で残します。少なくとも二つの100mm皿を一つの実験に必要とされます。 </li><li>ピペッティングによりセンダイウイルス感染細胞を回収し、15mlのコニカルチューブに転送し、室温で5分間、200×gで遠心します。 </li><li>上清を除去し、1mlの新鮮なKBM502媒体を追加します。 CEの数を数えます血球計数器を使用してLLSは、100 mmの基底膜マトリックス被覆に100 mmのステップ4.2で作製した料理やプレート1×10 5〜1×10 6細胞（10ml中の新鮮なmTeSR媒体）から基底膜マトリックス溶液を除去料理。最後に、コロニーは簡単にピックアップされたプレートを使用しています。 </li><li> 5％-CO 2インキュベーターO / Nで37℃で皿をインキュベートします。 </li><li>一日おきにmTeSR媒体を肉mlの10に培地を変更します。 注：約20〜30日、感染後、密接に胚性幹細胞（ESC）の似ているコロニーが表示されます。 </li></ol> 5. T細胞由来のiPS細胞を展開<ol><li> 4℃で（この研究で使用される特定のブランドのための材料のリストを参照）マトリックスゲルを解凍し、DMEM / F12培地10mlの0.2 mlで溶解することにより基底膜マトリックス溶液を調製します。 </li><li>プレート1 6ウェルプレート中でウェルあたり基底膜マトリックス溶液のミリリットルとで室温で残しますコロニーを選ぶ前に少なくとも30分。 </li><li>ウェルあたりmTeSR培地20μlを96ウェルプレートを埋めます。 </li><li> 20μlのピペットを用いて実体顕微鏡下のESCに似たコロニーを選択します。 注： 図1（b）に示すように、ESCのと性IPSCのコロニーをラウンドとフラットです。 </li><li>転送は、ステップ5.3でmTeSR媒体で満たされた96ウェルプレートにコロニーを選択しました。 </li><li> 200μlのピペットを用いて実体顕微鏡下で小さな塊にコロニーを破壊するために上下にピペッティングし、慎重に各ウェルにmTeSRの培地200μlを加え、。 5.2によく準備から基底膜マトリックス溶液を除去し、ウェルあたりmTeSR培地2mlで基底膜マトリックスでコーティングした6ウェルプレートの1ウェルに各コロニーを置きます。 </li><li>一日おきにmTeSR媒体を肉mlの2にメディアを変更します。 </li></ol> 6. T細胞由来のiPS細胞を維持 T細胞由来のiPS細胞を維持することができます人間性IPSCとヒトESCと同様の技術を使用して格納されています。 iPS細胞コロニーが成長したら、同じ手順を繰り返すことによって、より大きな皿にそれらを展開します。 <ol><li> 1〜2日ごとにmTeSR媒体を変更します。 </li><li>コロニーは、細胞は、製造業者の説明書に従って、ヒトiPS細胞およびヒトESCの解離溶液を使用して、毎5~7日経過コンフルエントになったとき。 </li></ol>

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著者らは、開示するいかなる競合や利害を持っていません。

We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

性IPSCは、再生医療1-3のための細胞の画期的な供給源として大きな注目を集めています。現在までに、iPS細胞を生成するための多様な方法が4,5報告されています。これらの中でも、ヒトT細胞から生成されたiPS細胞のための細胞採取6~8の低侵襲法の特に注目されています。さらに、T細胞由来のiPS細胞は、T細胞受容体（TCR）遺伝子の再配列を含み、したがって抗原特異的T細胞9,10の源です。そのため、安全性T細胞由来のiPS細胞を生成する再生医療の進展のために有用です。 この方法は、予測不可能な病原性の危険性を減少させるという概念に基づいています。性IPSCの安全な臨床応用のためには、病原体11への暴露の危険性を低減することが重要です。以前に多能性幹細胞の多くの培養系において、ウシ胎児血清およびフィーダー細胞を、必須の試薬として使用されています12秒 。 iPS細胞の生成は、予測不可能な病原性の危険性を低減するが、培養系からのこれらの試薬の両方を除去することが好ましいです。 さらに、この方法は、患者からの浸潤性細胞のサンプリングおよびフィーダー細胞の面倒な準備を回避するという利点を有します。 T細胞由来のiPS細胞は、既に疾患研究13,14に首尾よく使用されているため、この方法も適用可能であり、患者の疾患特異的iPS細胞を生成するために有用です。 遺伝子車両としてセンダイウイルス（SeVの）ベクターを用いて、T細胞の再プログラミング方法の中でも、高効率7,16でiPS細胞を生成することができる方法です。センダイウイルスは、一本鎖RNAウイルスであり、複製のためのDNAの相を必要としないため、また、iPS細胞の生成におけるその使用は、宿主ゲノム17-19を破壊が回避されます。したがって、我々は血清FRにおけるヒト末梢T細胞からiPS細胞を生成するためのプロトコルを確立していますEEおよびマトリゲル、mTeSR媒体の組み合わせを使用して無フィーダー条件、およびセンダイウイルスベクターです。

<table><tbody><tr><td>Ficoll-Paque PREMIUM</td><td>GE Healthcare</td><td>17-5442-02</td><td></td></tr><tr><td>Purified NA/LE mouse anti-human CD3</td><td>BD Pharmingen</td><td>555336</td><td></td></tr><tr><td>Bovine albumin fraction V solution</td><td>Gibco</td><td>15260-037</td><td></td></tr><tr><td>mTeSR1 medium kit</td><td>STEM CELL</td><td>5850</td><td>Warm at room temperature before use</td></tr><tr><td>Dissociation Solution</td><td>ReproCELL</td><td>RCHETP002</td><td></td></tr><tr><td>D-PBS(&amp;ndash;)</td><td>Wako</td><td>045-29795</td><td></td></tr><tr><td>SeV Vector kit CytoTune-iPS ver.1.0</td><td>DNAVEC</td><td>DV-0303c</td><td>Thaw on ice before use</td></tr><tr><td>100-mm tissue culture dish</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>96-well tissue culture plate</td><td>Falcon</td><td>353078</td><td></td></tr><tr><td>6-well tissue culture plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>15&amp;#160;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>188271</td><td></td></tr><tr><td>50&amp;#160;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr></tbody></table>

generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions

最近では、性IPSCは、再生医療のための細胞の新しいソースとして注目を集めています。 iPS細胞を生成するための初期の方法は侵襲的生検および導入遺伝子のレトロウイルスゲノムに挿入して得られた皮膚線維芽細胞に依存していましたが、これらの欠点を回避するために多くの努力がなされてきました。ヒト末梢T細胞がiPS細胞を生成するためのユニークな細胞源です。 T細胞由来のiPS細胞は、T細胞受容体（TCR）遺伝子の再配列を含み、抗原特異的T細胞の供給源です。さらに、ゲノム中のT細胞受容体の再配置は、個々の細胞株を標識し、移植したドナー細胞とを区別する可能性を有します。性IPSCの安全な臨床応用のためには、有害な薬剤に新たに生成されたiPS細胞を曝露する危険性を最小限にすることが重要です。ウシ胎児血清およびフィーダー細胞は、多能性幹細胞培養のために必須であったが、リスクを軽減するために、培養システムから削除することが好ましいです。予測不可能な病原性の。これに対処するために、我々は、未定義の病原体へのiPS細胞を曝露する危険性を低減するためにセンダイウイルスを用いて、ヒト末梢T細胞からiPS細胞を生成するためのプロトコルを確立しました。センダイウイルスを処理することは、適切なバイオセーフティーレベルと機器が必要ですが、センダイウイルスに感染はゲノム挿入せずに、まだ高効率でT細胞を活性化しました。このプロトコルでは、我々は、活性化T細胞培養およびセンダイウイルスの組み合わせを使用して無フィーダー条件でヒト末梢T細胞からのiPS細胞の生成を実証します。

<html>このプロトコルを使用して、ユーザは安定したヒト末梢T細胞からiPS細胞を生成することができます。センダイウイルスおよびマトリゲルを有するT細胞からのiPS細胞の生成は、約0.002％を示した-いくつかのドナー例とのSeVの間での細胞のリプログラミングの効率の0.005％は、いくつかの通路16の後には検出されなかった。図1（a）は、T細胞由来を生成するためのプロトコルの概略図を示します。マトリゲルとmTeSR媒体を用いた無フィーダー条件で性IPSC。センダイウイルス感染後20〜30日の周りに、iPS細胞のコロニーをそれらのESCコロニー様形態（ 図1B）によって認識されます。免疫蛍光染色は、無フィーダー条件（ 図1C）の下で生成されたT細胞由来のiPS細胞に典型的な多能性細胞マーカー（NANOG、OCT3 / 4、SSEA4、TRA-1-60、およびTRA-1-81）の発現を明らかにしました。 &quot;幅=&quot; 700 &quot;/&gt; 図1（A）：この研究では、フィーダーフリー条件下での再プログラミングT細胞のためのプロトコルの概略図。 （B）：典型的なESCのようなiPS細胞コロニーフィーダーフリーの条件下で採血後27日目に。 （C）：T細胞由来のiPS細胞でALP染色および多能性および表面マーカーの免疫蛍光染色（NANOG、OCT3 / 4、SSEA4、TRA-1-60、およびTRA-1-81）は、フィーダーを含まない条件下で生成<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">してくださいこの図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

フィーダーフリー条件にセンダイウイルスを用いてヒト末梢T細胞から人工多能性幹細胞の生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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Developmental Biology

11.9K ビュー.  Keio University School of Medicine. This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

ビデオ: Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

発生生物学

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

非統合ソームプラスミドを用いて凍結軟膜から誘導多能性幹細胞の生成

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

ヒトメラノーマ腫瘍浸潤リンパ球から誘導多能性幹細胞の生成

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

センダイウイルスや遠心分離を用いて血液細胞から誘導多能性幹細胞の生成

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

エピソームベクトルを用いたヒト末梢血単核細胞からの統合フリー人工多能性幹細胞の生成

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

末梢単核細胞からの誘導神経幹細胞の生成と移植研究におけるドーパミン作動性ニューロン前駆体への分化

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

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 ...