Development of this protocol was supported by the Advancing a Healthier Wisconsin and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

注：患者、および/またはその保護者は、研究を承認した治験審査委員会に参加するためにインフォームドコンセントを与える必要があります。 1.バッファとメディアの準備<ol><li>洗浄緩衝液：リン酸緩衝食塩水（PBS）の99 mlの100×ペニシリン/ストレプトマイシン溶液を1ml（100 U / mlペニシリン+ストレプトマイシン100μg/ mlの）を追加し、洗浄緩衝液100mlを調製する。 </li><li>尿中前駆細胞（UPC）ミディアム：ケラチノサイト無血清（KSF）ミディアム+前駆細胞培地を等量混ぜ、UPC媒体を作製した。 <ol><li> KSF中：上皮成長因子の5 ng / mlの（EGF）、ウシ下垂体抽出物の50 ng / mlの（BPE）、コレラ毒素の30 ng / mlの、およびペニシリン/ストレプトマイシン溶液（100 Uを追加し、ケラチノサイト培地を調製するために/ mlペニシリン、500ミリリットルKSF培地に100μg/ mlのストレプトマイシン）。 </li><li>前駆細胞培地：10％FBSでハム-F12培地とサプリメントの四分の一を含むDMEMの4分の3の追加、0.4 / mlのヒドロコルチゾン、0.1nMのコレラ毒素、5 / mlのインスリン、1.8×10 -4 Mのアデニン/ mlのトランスフェリン、2をx 10 -9 Mのトリヨードサイロニン、10ng / mlのEGF、およびペニシリン/ストレプトマイシン溶液を5μg。 </li></ol></li><li>のhES培地：/ 20％KO血清代替（K-SR）を追加し1×MEM非必須アミノ酸、2mMのL-グルタミン、100μMのβメルカプトエタノール、20ng / mlのbFGFおよびペニシリン/ストレプトマイシン400 mlのDMEM F12培地。 </li></ol> 2.尿サンプルコレクション<ol><li>尿の十分な量（〜30〜40ミリリットル）を収集することができることを保証するために採尿に30分前に流体を飲むように患者に指示します。 </li><li>患者および/またはその保護以下の項目を含む尿採取キットを与える：（1）滅菌またはクリーンキャッチ尿サンプルを入手する方法についての説明書、（2）湿った抗菌toilettes、及び（3）を100mlの滅菌標本コレクションカップ。サンプルを収集するために患者に指示します。 </li><li>直ちに氷上に尿サンプルを配置し、laboraに移転クーラーでトーリー。すぐに細胞単離のための尿を処理します。遅延が避けられない場合には、細胞の生存率の損失を最小限に抑えて、最大4時間氷上で試料を保存する。 </li></ol> 3.単離と尿細胞の拡大注：BSL2生物学的安全キャビネット内無菌条件下で、次の手順を実行します。 <ol><li>滅菌ピペットを用いて、転送尿サンプルを50ml遠心管に無菌である。 RTで10分間、400×gで遠心分離する。 </li><li>チューブ内の1ミリリットルを残して上清を吸引。細胞ペレットを乱さないように注意してください。 </li><li>再懸濁洗浄緩衝液7mlの中に複数の管からペレットを組み合わせて、RTで10分間、400×gで遠心分離を繰り返し。 </li><li>慎重に細胞ペレット及び上清〜0.2〜0.5ミリリットルを残して、上清を吸引除去する。尿中前駆細胞（UPC）培地2〜3mlの中で再懸濁し、国連の4〜6ウェルにサスペンションを転送コー​​トした24ウエルプレート。 </li><li> 72時間後、ウェル当たり新鮮UPC培地の0.5ミリリットルと文化を補完し、2〜3日ごとに後の培地に変更します。プレートに付着していない赤血球と扁平上皮細胞を培地変更で削除されます。 </li><li> 4-6日後の培養を監視します。 注：小2-4細胞コロニーが現れ始めます。細胞は、それぞれタイプIまたは腎上皮のタイプII（RE）のセル3を表す丸めまたは伸長される。 </li><li>細胞が表示されたら、彼らは急速な拡大期を受けることになるので、2〜3日毎に培地を変更します。 </li><li>細胞がプレーティング後9-15日を中心に、80〜90％のコンフルエンスに到達すると、さらなる拡大のために24ウェルプレートの2-4ウェル上に細胞（15,000-18,000細胞/ cm 2）を解離して通過。細胞継代1（P1）としてこれをマークします。 </li><li>フローサイトメトリー分析を介して系統仕様の尿細胞を特徴づける、immunohistochemiCalの染色または逆転写酵素 - ポリメラーゼ連鎖反応（RT-PCR）を介し。 </li><li>長期間の凍結保存のために（DMEM、10％血清および10％DMSOを補った）孤立した尿細胞（セクション4）を再プログラムまたは凍結培地にそれらを凍結する。 </li></ol>センダイウイルスを使用する4.尿細胞リプログラミング注：トランスフェクションエージェントを操作しながら適切な取り扱いとPPE（個人用保護具）の使用が推奨されます。 BSL2生物学的安全キャビネット内無菌状態で、次の手順を実行します。薬剤および/またはトランスフェクトされた細胞をトランスフェクトする適切な処分は、環境と健康の危険のリスクを回避することをお勧めします。以下に詳述するように、尿細胞の再プログラミングについては、メーカーのフィーダー依存プロトコルへの変更を加えて仙台リプログラミングキットを使用しています。 <ol><li>センダイウイルスを用いた高効率な再プログラミングを確保するために、使用が急速である尿細胞の1-5通路LYは分裂。細胞が十分に複製または老化にならない場合は、彼らが効率的に再プログラムしないので、それらを捨てる。 </li><li> 6ウェルプレートの2つのウェルにおける種子ウェル当たり60,000個の細胞。マーク·デイ-2としてこの。細胞播種密度を調整し（5×10 4から 9×10 4個の細胞をウェルあたり）0日によって80〜90％コンフルエンスに到達するために必要である。 </li><li> 0日目（細胞を播種後48時間）で、4 OSKM因子のそれぞれを含む仙台リプログラミングベクター（SeVの）を調製。予め温めUPC培地1mlにベクトルの各々を加える。尿細胞を再プログラミングするための十分である、1〜1.5のMOI（5〜7.5×10 5 CIU）を使用します。メディアを吸引し、ゆっくりと尿細胞の1ウェルに1ミリリットルUPC +のSeVを追加します。形質導入ネガティブコントロールとして第2のウェルを使用してください。 </li><li> 1日目に、新鮮なUPC媒体とUPC + SeVの媒体を交換してください。いくつかの細胞死は、ウイルス細胞毒性によるものでは24時間後に見られている。 </li><li>再プログラムCLONの効率に応じて、Eの形成とコンパクトな形態は、6日目または7まで毎日培地を変え続ける。 </li><li>前に形質導入した細胞（5日目または6）、マイトマイシンC（3時間、10μg/ ml）を0.1に5×10 4細胞/ cm 2の密度で処理された、MEFのメッキをMEFフィーダープレートを準備の解離の一日％のゼラチンは100ミリメートル2培養皿をコーティングした。 </li><li>次の日（6日目または7）、0.25％トリプシンで細胞を解離UPC培地プレートに5×10 4細胞を再懸濁する- 2×10 5細胞/ 100のmm 2のMEFフィーダープレート。 </li><li>次の日は、hES培地に切り替えて、毎日培地を変更します。形質転換細胞を監視するために、顕微鏡下で細胞を観察します。 注：細胞は、特徴的な敷石状形態を有するクローンの凝集体を形成し、細胞質の比率（12-18日後に形質導入​​）に高い核を有する。 </li><li>形質導入後2〜3週間以内に、コロニーをピックアップし、CLONのための新しいプレートに移すアル展開。 </li><li> 37℃で1時間、TRA-1-81抗体で細胞をインキュベートすることによって、IPSCクローンを選択するための生細胞染色を行う CO 2インキュベーター中で37℃で1時間、特定の蛍光色素結合二次抗体との対比染色に続いてCO 2インキュベーター内でC°。 </li><li>小さな等しい大きさの小片（クローンあたり4-6個）にクロスハッチ大きなTRA-1-81陽性のIPSCクローンに26 G 1.5インチの針を使用してください。 </li><li>マトリゲル（10μgの/ cm 2程度）の各ウェルに複数のクローンから10-20クロスハッチング部分をピックアップし、転送するために滅菌ピペットチップを使用して、hES培地で24ウェルプレートをコーティングさ。 注：単一のウェルに個々にメッキ単一のクローンからの小品が展開または多能性を維持していない。 </li><li>再プログラムされたクローンは、マトリゲル表面に取り付けられた後のIPSC媒体へのhESからの変化24〜48時間。 </li><li>マトリ被覆プレート上のメンテナンス時に、手動でSCRAPE-オフあらゆる分化した細胞または完全に再プログラムし、多能性ジストロフィーIPSC（MD-IPSC）のクローンを濃縮するためにピペットチップを用いてMEFフィーダー細胞を汚染する。 </li><li>個々のクローンは、〜100μmのサイズに達した後、3分間の0.48 mMのEDTA（エチレンジアミン四酢酸）溶液を用いて解離および5μMY27632補充したIPSC培地に懸濁MD-iPS細胞の小細胞凝集体（、RhOなどキナーゼ阻害剤をめっきすることによってクローンを拡大する）新鮮なマトリゲル上で（を10μg/ cm 2）をコーティングした12ウェルプレート。毎日のIPSC媒体を変更します。 </li><li>各クローンの完全な再プログラミングはOSKM遺伝子および多能性マーカー（のOct3 / 4、TRA-1-81）の免疫組織化学染色の両方の遺伝子発現解析によって決定することができる。多能性の可能性の厳格な評価は完全に再プログラムIPSC線が三胚葉に分化することができる確認するために用いられるべきである。これは、胚様体（EB）形成および分化アッセイによって、またはターどちら達成することができる血腫形成アッセイ。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

性IPSCを使用して、心血管疾患のモデリングは、遺伝的寄与4-6を理解するための一般的なアプローチになってきている。患者から細胞サンプルを取得するいくつかの予期せぬ困難、特に幼い子供たちは、そのような尿収集などの非侵襲的アプローチのオプションを提供することによって回避することができる。この若い患者集団では、再プログラミングのための十分な尿細胞を得るの?...

心筋症は、デュシェンヌ型とベッカー型筋ジストロフィー（MD）患者の死亡原因の第2位である。 X連鎖ジストロフィン遺伝子の変異が1で発生するものの：3500男性の出生、非常に少ない、プログレッシブ心筋の損傷につながる分子や細胞のイベントについて知られている。筋ジストロフィー患者由来のヒト誘導多能性幹細胞は、基礎疾患の機構を研究するために、薬剤は、1,2のスクリーニングに使用するための新規なツールとして浮上している。 皮膚生検または血液サンプルの予想不快感は、研究参加の同意を与えるために若い患者及び/またはその保護者を説得することができる。尿サンプルは、再プログラミング方法に修正可能である体細胞の非侵襲的な供給源である。我々は最近、筋ジストロフィー患者から採取した尿細胞は培養することができることを示しかつ効率的に山中因子とレトロウイルス形質導入を用いて、iPS細胞（に再プログラムしているあるOct3 / 4、Sox2の、Klf4及びc-Myc; OSKM）1。レトロウイルス遺伝子送達の欠点は、宿主染色体への初期化遺伝子のランダムな統合です。この制限を克服するために、我々は、尿細胞再プログラミングのための非組み込み型センダイウイルスを使用している。 このプロトコルは、さらなる研究のための心筋細胞または他の細胞型に分化させることができる筋ジストロフィー患者から単離された尿細胞のセンダイウイルス再プログラミングについて詳しく説明します。このプロトコルは、他の患者の特定の疾患に適合させることができる。

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1144px;">List of Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 Oz. Specimen Cup with Lid</td>
			<td>STL Medical Supply</td>
			<td>M9AMSAS340</td>
			<td>For Urine Sample Collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round Glass Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-81</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CytoTune-iPS Sendai Reprogramming Kit</td>
			<td>Life Technologies</td>
			<td>A1378-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen-Strep w/o Glutamine&#160;</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/o Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>0050129SA</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 (1:1)</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene, 1:5000&#160;</td>
			<td>Life Technologies</td>
			<td>15040066</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bFGF (10 &#181;g)</td>
			<td>Life Technologies</td>
			<td>13256-029</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counter Cartridges</td>
			<td>Life Technologies</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum (500 mL Bottle)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recovery Cell Culture Freezing Medium</td>
			<td>Life Technologies</td>
			<td>12648-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, High Glucose, Glutamax</td>
			<td>Life Technologies</td>
			<td>10566-016</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ham&#39;s F-12 Nutrient Mix&#160;</td>
			<td>Life Technologies</td>
			<td>11765-054</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGF Recombinant Human Protein, Liquid Form</td>
			<td>Life Technologies</td>
			<td>PHG0311L</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin, Human Recombinant, Zinc Soln</td>
			<td>Life Technologies</td>
			<td>12585-014</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TeSR-E8</td>
			<td>Stem Cell Technologies</td>
			<td>5940</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ROCK Inhibitor (Y-27632)</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>hESC Qualified Matrix, LVED Free</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>170-8890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DreamTaq Green PCR Master Mix (2X)</td>
			<td>Thermo-Scientific</td>
			<td>K1081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS-Qualified</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ADENINE BIOREAGENT</td>
			<td>Sigma Aldrich</td>
			<td>A2786-5G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHOLERA TOXIN FROM VIBRIO CHOLERAE</td>
			<td>Sigma Aldrich</td>
			<td>C8052-.5MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HYDROCORTISONE</td>
			<td>Sigma Aldrich</td>
			<td>H0888-1G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HOLO-TRANSFERRIN FROM HUMAN</td>
			<td>Sigma Aldrich</td>
			<td>T0665-50MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,3&#39;,5-TRIIODO-L-THYRONINE SODIUM SALT&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T6397-100MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-3598</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluoromount Aquous mounting medium</td>
			<td>Sigma Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% Paraformaldehyde</td>
			<td>Alfa-Aesar</td>
			<td>43368</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">List of Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD44 - labelled with FITC&#160;</td>
			<td>BD Biosciences</td>
			<td>560977</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD146 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD73 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-&#945;-smooth muscle actin</td>
			<td>Sigma Aldrich</td>
			<td>A2547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-Vimentin</td>
			<td>Abcam</td>
			<td>ab8978-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Anti - TRA-1-81</td>
			<td>Life Technologies</td>
			<td>411100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti Oct 3/4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-9081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:415px;">Mouse Anti Dystrophin</td>
			<td>Leica Biosystems</td>
			<td>NCL-DYSB</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells

異栄養性心筋症は、筋ジストロフィーのあまり理解結果である。筋ジストロフィーの患者から誘導された多能性幹細胞（iPS細胞）を生成するインビトロ疾患モデル系のための貴重な細胞源であり、薬剤スクリーニング研究のために使用することができる。患者由来の尿細胞はジストロフィー性心筋1をモデル化するために、誘導多能性幹細胞への成功した再プログラミングに使用されてきた。ベクター系を統合する安全性の問題に対処する、我々は、筋ジストロフィーの患者から採取した尿細胞に山中因子の形質導入のための非組み込み型センダイウイルスベクターを使用してプロトコルを提示する。このプロトコルは、2〜3週間以内に完全に再プログラムされたクローンを生成します。多能性細胞は、流路13によりベクターフリーです。これらの異栄養性IPSCは、心筋細胞に分化し、病気のメカニズムを研究または薬物スクリーニングするために使用することができる。

（それぞれ97.37パーセント、97.09パーセント、および97.3％; 図1Aおよび図1B）は、ヒト尿から単離されたほとんどの前駆細胞は、CD44、CD73、およびCD146などの尿路上皮前駆細胞および周皮細胞のマーカーに対して陽性である。これらの細胞は、α平滑筋アクチン、およびビメンチン（ 図1B）のような他の間葉系マーカーを発現した。サイトケラチン-7（CK-7）?...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells at JoVE.com

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

筋ジストロフィー患者から人工多能性幹細胞の生成：尿由来細胞の効率的な統合·フリーリプログラミング

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

generation-induced-pluripotent-stem-cells-from-muscular-dystrophy

Research

JoVE Journal

Medicine

11.1K ビュー.  Medical College of Wisconsin. This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction. 

ビデオ: Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前立腺癌患者の発生は、循環腫瘍細胞からの異種移植モデルを派生します

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

C2C12筋芽細胞における筋強直性ジストロフィー1のモデル化

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

中リコンビナーゼ媒介カセット交換による組換えヒト多能性幹細胞の迅速かつ効率的な生成<em&gt; AAVS1</em&gt;軌跡

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

フォルスコリン誘発性腸オルガノイドに腫れ：<em&gt;インビトロ</em&gt;アッセイ嚢胞性線維症患者における薬物反応を評価するための

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

によるエクソン介在物評価スプライスの SMA 患者の繊維芽細胞でスイッチング アンチセンスオリゴヌクレオチド

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

ひと臍帯血由来 cd34 陽性造血幹細胞の体外増幅+細胞を用いたバルプロ酸

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

出生後初期の発達におけるエメリー・ドレイフス型筋ジストロフィーのマウスモデルからの単一の筋繊維の分離と培養

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...