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 border="0">
	<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>KBM502 medium</td>
			<td>KOHJIN BIO</td>
			<td>16025020</td>
			<td>Warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4&#8451; overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</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(&#8211;)</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>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</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://www.jove.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

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, ...

発生生物学

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

髪の由来ケラチノサイトを使用した統合フリーヒト誘導多能性幹細胞の生成

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

ヒト多能性幹細胞からメラノサイトのフィーダーフリーの導出

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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 ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

ヒト多能性幹細胞から肝細胞様細胞の定義され、スケーラブルな世代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

末梢血由来ヒト誘導多能性幹細胞からの軟骨細胞の分化

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

標準化され、再現性のある前脳型人間から脳オルガノイドの生成誘導多能性幹細胞

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

臍帯血由来から 3 D 肌 Organoid の生成誘導多能性幹細胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...