この作品は、チャールズ·フッド財団からの医学、母子保健研究賞のイェール大学によって資金を供給された。

1。初期化因子を発現するレトロウイルスによるプログラミング<ol><li>ヒト線維芽細胞は線維芽細胞の培地（10％ペン/連鎖球菌を含むDMEM中でFBS）で培養されています。 </li><li> 6ウェルプレートの1ウェルに感染する前に、一日は、プレート1×10 5、ヒト線維芽細胞。 </li><li>死んだ細胞を除去し、新鮮な線維芽細胞の培地2mlを追加するには、培地を吸引除去する。 5μg/ mlの最終濃度で硫酸プロタミンを追加します。 </li><li>慎重に感染多重度（MOI）5に対応する各GFP発現ウイルスの適切な量を追加します。 </li><li>感染後一日、ウイルス上清を除去し、2 mlの線維芽細胞の培地を追加し、2 mlのPBSで3回洗浄する。 </li><li>感染3日後、GFPの蛍光を確認し、2 mlの線維芽細胞培地でウェルを補充する。 </li><li>四日間の上に線維芽細胞培地中で照射したマウス胚性線維芽細胞（MEF）フィーダー細胞の感染は、プレート1×10 4 / cm 2とした後0.1％ゼラチンでコーティングされた10cmのペトリ皿。 37℃で一晩インキュベートします。 </li><li>五日、感染後、1ミリリットル0.05パーセントtypsin / EDTA、5分間37℃、および200 gで5分間遠心分離に感染したヒト線維芽細胞を切り離します。培地を吸引除去し、線維芽細胞の培地10mlので細胞を再懸濁します。プレコート10cmのプレートに細胞を移す。 </li><li> 24時間後、hESCの培地（20％ノックアウト血清代替物、DMEM/F12、0.1ミリリットル非必須アミノ酸、4 ng / mlのbFGFを、ペン/球菌/グルタミン酸、β-merceptoethanol）で培地を交換してください。毎日の培地を変更します。 ESCのようなコロニーは感染後20から27日で表示を開始します。 </li></ol> 2。 iPSCsの分離と拡張<ol><li>蛍光顕微鏡下で、ヒトES細胞に似た形態を示しています。植民地におけるGFP蛍光の有無を確認してください。 </li><li> 10μlのピペットを用いて、個々のIPSCのコロニーをピックアップし、ゼラチンおよびMEF-CoAの1つのウェルに配置しますテッド12ウェルプレートとhESCの培地を添加した。毎日の培地を変更します。 </li><li>継代については、DMEM/F12 1mlでプレートを洗浄し、コラゲナーゼの0.5ミリリットルを追加し、37℃で10分間インキュベート℃、 </li><li> DMEM/F12で細胞を2回洗浄します。 </li><li>新鮮なhESCの培地2mlを追加します。セルリフターを使用して、小さな部分にコロニーを破壊し、プレートから残りの細胞を切り離します。 </li><li>ゼラチンとMEF-コーティングされた6ウェルプレートの1ウェルにコロニーの再懸濁の部分を転送します。 </li></ol> 3。多能性マーカーの免疫蛍光分析<ol><li> PBSで細胞を3回洗浄し、室温で20分間、4％パラホルムアルデヒドで固定します。 </li><li>静かにPBSで細胞を3回洗浄し、30分間PBSでトリトンX-100 0.2％透過性。 </li><li> 2時間のPBSで3％BSAで細胞をインキュベートすることにより、非特異的結合をブロックします。 </li><li> 4℃で一晩一次抗体で細胞をインキュベートします。 </li> &lt;LI&gt; PBSで細胞を3回洗浄し、光から遮蔽し、室温で1時間の特定の二次抗体で細胞をインキュベートします。 </li><li>室温で5分間インキュベートし、続いて最後の洗浄時にPBSおよびアドオンDAPIで細胞を3回洗浄します。 </li><li>蛍光顕微鏡による染色を検出します。 </li></ol> 4。多能性マーカーの定量的リアルタイムPCRアッセイ<ol><li>キアゲン社のRNeasyキットを用いて、ヒト線維芽細胞由来のヒト性IPSCからトータルRNAを分離します。 </li><li> Superscript II逆転写酵素を用いた第一鎖cDNAを合成する。 </li><li>以前に報告されたプライマーを用いて、多能性の遺伝子を検出するために定量PCRを実行します。6 </li></ol> 5。代表的な結果<ol><li> プログラミング時の形態変化 我々は、OCT4、SOX2、KLF4およびMYCを運ぶレトロウイルスのカクテルで、ヒト線維芽細胞BJ1とデトロイト551感染と（ 図1）プログラミング時の形態学的変化を検出することができました。二十一日、感染後、我々は彼らのhESCのような形態で小さなIPSCコロニーを認識しています。さらに、我々はGFPの蛍光により性IPSCを認識しています。そのようなESCと性IPSCとして多能性幹細胞、プロウイルス遺伝子発現7-9を抑制する分子機構を表現しています。当社独自のレトロウイルスベクターは、レトロウイルスLTRで遺伝子を再プログラミングと一緒にGFPを表現しています。したがって、継続的にGFPを発現する細胞は、プロウイルス遺伝子サイレンシングすることなく導入遺伝子を発現するように考えられている。多能性分子ネットワークを取得忠実に再プログラムIPSCコロニーはGFP発現（ 図2）10の不在を示しています。 </li><li> 人間のiPSCsの多能性のキャラクタリゼーション 我々は、TRA-1-81、TRA-1-60、SSEA-4、SSEA-3、OCT4とNANOG抗体を用いた免疫組織化学を経由してデトロイト-551線維芽細胞由来コロニーを分析した（ <stroNG&gt;表2）。首尾よく再プログラム性IPSCは、これらのマーカー（ 図3A）のすべてを表現しています。また、定量的RT-PCR分析を介して遺伝子発現を解析した。我々は、OCT4、SOX2、KLF4、MYCとNANOGの発現が有意に親の線維芽細胞と比較しますが、H9ヒトES細胞（ 図3B）のそれに見合った増加したことを観察した。 </li></ol><img alt="図1" src="/files/ftp_upload/3804/3804fig1.jpg" /> 図1。レトロウイルスに感染したヒト線維芽細胞の形態変化（AD）初期化因子に感染したデトロイト-551線維芽細胞からのコロニーでプログレッシブ形態変化。 5日目（）、10日目（B）、14日目（C）、21日（D）。細胞は21日後hESCのような形態を示しています。 <img alt="図2" src="/files/ftp_upload/3804/3804fig2.jpg" /> 図2。リプログラミングを受けている細胞の代表的GFP蛍光発現。 </strong&gt; BJとデトロイト551線維芽細胞は、4つの初期化因子4を発現するレトロウイルスに感染し、4週間hESCの培地中でインキュベートした。 BJ繊維芽細胞（A、B）とデトロイト551（C、D）は、形態学的類似の表示されます。 21日から、GFP陰性のコロニーは10善意性IPSCを表現する、形成するために開始します。 （E、F）適切なリプログラミングを受けていないデトロイト551細胞の形質転換を示しています。位相コントラストビューの下に（A、C、E）のコロニー。 （B、D）が正しくGFPサイレンシングを示す細胞を再プログラム。形質転換コロニーから（F）明るいGFP発現。 <img alt="図3" src="/files/ftp_upload/3804/3804fig3.jpg" /> 図3。人間の人工多能性幹細胞のキャラクタリゼーション。（a）ヒト551-IPS-K1細胞のコロニーは、多能性細胞に共通のマーカーを発現する。 DAPI染色は、フィールドごとの合計のセルの内容を示しています。 OCT4、SOX2の発現のために（B）定量リアルタイムPCR（RT-QPCR）、KLF4、MYC親のfのibroblast、551-IPS-K1の性IPSC、およびH9ヒト胚性幹細胞（ヒトES細胞）。データは、β-アクチンのハウスキーピング遺伝子に対して正規化し、4親の線維芽細胞での発現レベルを基準にプロットした。

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<p class="jove_content"&gt;私たちは、開示することは何もありません。</p

性IPSCの4つの転写因子の発現は、ヒト線維芽細胞を再プログラムします。多くの試みが臨床的に安全性IPSCを生成するために、非積分または非遺伝的アプローチを用いてヒトの性IPSCを生成するために行われました。これまでのところ、これらのメソッドは非常に低い効率を示し、再現性11月14日を向上させるためにさらに最適化する必要があります。レトロまたはレンチウイルスの方法は容易に導出し、ウイルスの統合に起因する安全性の問題にあまり依存しているin vitroでの疾患モデルで人間のための性IPSCを適用するために使用されます。ここで説明する方法を再プログラミングすることは、ヒトのiPSCsの効率的な導出のために利用可能です。人間のiPSCsの選択は、主にヒトESCに似ているコロニー形態に基づいています。もっと重要なのは、私たちの方法は、多能性幹細胞7,15でレト ​​ロウイルスの長い末端反復配列（LTR）のサイレンシングの機能を利用しています。このプロトコルで使用されているレトロウイルスベクターは、リプログラミングにリンクされているGFP遺伝子を含む内部リボソーム侵入配列（IRES）と5を介しての要因。 GFPの発現はプロウイルスLTRにより駆動される。これらのウイルスに感染した線維芽細胞は最初に明るいGFP発現を示しています。一度完全に再プログラム、性IPSCは、蛍光顕微鏡下で容易に可視化されるGFPの発現を、失うことになります。このプロトコルでは、我々は、胎児や新生児線維芽細胞（デトロイト551とBJ1）からiPSCsの生成を説明します。しかし、このレトロウイルスベクターは、通常成人線維芽細胞からの性IPSCと同様にメンデルや複雑な疾患4,16,17患者のさまざまなを生成するために使用されています。 以前に我々は10を再プログラムする人間の体細胞中に細胞表面マーカーの変化を分析した。細胞表面マーカーの漸進的変化があります。線維芽細胞は、プログラミングの発現によって抑制されるCD13を発現する。細胞は、GFPとSSEA4を一緒に表現するためにプログラミングの開始を受けている。そして、彼らはexpressiを失うプロウイルスsilecingと急行の追加多能メーカーTRA1-60 10を介してGFPの上に。 TRA1-60の発現がよくTRA1-60を忠実に再プログラム性IPSCのマーカーであることを示唆し、GFPのサイレンシングとよく発達した奇形腫の形成と相関している。 GFPサイレンシングは、TRA1-60の発現のための代替マーカーであり、骨の折れる生細胞染色せずにIPSCコロニーの同定を可能にする。性IPSCのためのマーカーとしてGFP発現の損失を使用して、容易に、一貫性IPSCを分離します。プログラミングには経験を持たない細胞の科学者を食い止める。

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">hESC medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>80%</td>
			<td>Invitrogen</td>
			<td>11330057</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>20%</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>2 mM</td>
			<td>Invitrogen</td>
			<td>25030081</td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids (10 mM)</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>11140050</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol (14.3 M) or MTG</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>M-6250</td>
		</tr>
		<tr>
			<td>bFGF-2 10 &mu;g/ml</td>
			<td>4 ng/ml</td>
			<td>GIBCO/BRL</td>
			<td>GF003AF</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td colspan="4">Fiboblasts Medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
			<td>Invitrogen</td>
			<td>11965118</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
			<td>Invitrogen</td>
			<td>10407028</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
	</tbody>
</table>
Table 1. Culture Medium
<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab19857</td>
		</tr>
		<tr>
			<td>SSEA3</td>
			<td>1:100</td>
			<td>Milipore</td>
			<td>MAB4303</td>
		</tr>
		<tr>
			<td>SSEA4</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560218</td>
		</tr>
		<tr>
			<td>Tra-1-81</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560173</td>
		</tr>
		<tr>
			<td>Tra-1-60</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560174</td>
		</tr>
		<tr>
			<td>NANOG</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab21624</td>
		</tr>
		<tr>
			<td>Alexa-Flur 488</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa-Flur 555</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A21422</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>1:5000</td>
			<td>Invitrogen</td>
			<td>D1306</td>
		</tr>
		<tr>
			<td colspan="4">Plasmids</td>
		</tr>
		<tr>
			<td>pMIG-OCT4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17225</td>
		</tr>
		<tr>
			<td>pMIG-SOX2</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17226</td>
		</tr>
		<tr>
			<td>pMIG-KLF4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17227</td>
		</tr>
		<tr>
			<td>pMIG-MYC</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>18119</td>
		</tr>
		<tr>
			<td colspan="4">Other Materials</td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>1mg/ml</td>
			<td>Invitrogen</td>
			<td>17104019</td>
		</tr>
		<tr>
			<td>Gelatin, Porcine</td>
			<td>0.1%</td>
			<td>Sigma</td>
			<td>G 1890</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>0.2%</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>4%</td>
			<td>Sigma</td>
			<td>47608</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>3%</td>
			<td>American Bioanalytical</td>
			<td>AB01800</td>
		</tr>
		<tr>
			<td>MEF feeder cells</td>
			<td>&nbsp;</td>
			<td>Millipore</td>
			<td>PMEF-N</td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>&nbsp;</td>
			<td>Corning</td>
			<td>3008</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td colspan="4">Fluorescent microscopy: inverted microscope with GFP filter</td>
		</tr>
	</tbody>
</table>
Table 2. Reagents and equipment.

reprogramming human somatic cells into induced pluripotent stem cells (ipscs) using retroviral vector with gfp

ヒト胚性幹細胞（ヒトES細胞） のin vitro疾患モデルと再生医療1 のための多能性と非常に貴重な細胞の源である。それは以前にヒト体細胞は、4つの転写因子の異所性発現（Oct4の、レトロウイルスベクター、KLF4及び Myc）によって多能性を再プログラムすることができることを示して誘導多能性幹細胞（性IPSC）2-4になっています。ヒトES細胞と同様に、人間性IPSCは、多能性と自己細胞の潜在的な源である。ここでは、GFPを含有するレトロウイルスバックボーン4にクローン化された4初期化因子によるヒト線維芽細胞を再プログラムするためのプロトコルについて説明します。以下のプロトコルを使用して、我々は、ヒトESCを培養条件下で3〜4週間で人間性IPSCを生成します。人間のIPSCのコロニーが密接に形態の似ているヒトES細胞とレトロウイルスのジーンサイレンシングの結果、GFP蛍光の損失を表示します。蛍光microscoの下に機械的に隔離されたIPSCのコロニーPEは、ヒトES細胞と同様の方法で動作します。これらの細胞では、我々は、複数の多能性遺伝子と表面マーカーの発現を検出することができます。

Watch this Scientific Journal Video about Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP at JoVE.com

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

OCT4、SOX2、KLF4とMYCのレトロウイルス媒介性異所性発現を介してヒトの人工多能性幹細胞（性IPSC）を生成する方法が記載されている。 GFP発現に基づいて人間のIPSCコロニーを識別するための実用的な方法についても説明されています。

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 ...

reprogramming-human-somatic-cells-into-induced-pluripotent-stem-cells

Nanyang Technological University

Research

JoVE Journal

Biology

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

20.4K ビュー.  Yale School of Medicine. OCT4、SOX2、KLF4とMYCのレトロウイルス媒介性異所性発現を介してヒトの人工多能性幹細胞（性IPSC）を生成する方法が記載されている。 GFP発現に基づいて人間のIPSCコロニーを識別するための実用的な方法についても説明されています。

ビデオ: Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

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

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

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

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

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

生物学

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

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

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

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

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

オリゴデンドロサイト前駆体に胚性幹細胞の分化

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

大人の線維芽細胞から再プログラムヒト人工多能性幹細胞のコロニーを選択し、分離

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

培養ヒト胚および誘導多能性幹細胞に適したマウス胚性線維芽細胞の調製

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...