この作品は、WMに国立衛生研究所からの補助金（CA127574、CA107040とCA09071）によってサポートされていました

1。マウスから収穫の異種移植<ol><li>ダルベッコ改変イーグル培地（DMEM）10％ウシ胎児血清（FCS）、ペニシリン - ストレプトマイシン、200 U / mLのコラゲナーゼIV型、および0.6 U / mLのディスパーゼを添加した：細胞解離バッファーを準備します。 </li><li>最大直径1cm未満である皮下ヒト膵臓癌異種移植を保有する無胸腺（NU + / NU +）マウスは、二酸化炭素の窒息と頸椎脱臼により安楽死させています。 </li><li>皮下腫瘍は​​、鉗子とハサミを用いて鈍的切開によってマウスの脇腹から採取し、細胞解離緩衝液中で直後に配置されます。 </li><li>腫瘍の重量を測定し、腫瘍組織の1グラムあたり10 mLの細胞解離バッファーに戻されている。 </li></ol> 2。異種移植片からの単一細胞懸濁液を生成する<ol><li>無菌バイオセーフティーキャビネットでの作業、腫瘍は細胞解離緩衝液1 mLと100 × 20 ​​mmの培養皿に転送されます。腫瘍は、機械的に無菌のかみそりの刃やピンセットを使って刻まれている。腫瘍細胞懸濁液を5 mLの血清学的ピペットを通して10回トリチュレートしています。腫瘍の部分は、5 mLの血清学的ピペットを詰まらせないために十分小さくする必要があります。 </li><li>腫瘍細胞懸濁液は、細胞解離バッファー（塗りつぶされた50％未満）の残りのボリュームを含む50 mLコニカルチューブに移し、1分間最大速度でボルテックスしています。 </li><li>腫瘍細胞懸濁液は、℃で2時間1分毎に20分間断続的にボルテックスしながら37℃でインキュベートされる。 </li></ol> 3。組織破片及び死細胞の細胞懸濁液を使い果たす<ol><li> 2時間のインキュベーション後に細胞懸濁液は、1分間の最大速度でボルテックスしています。 </li><li>細胞懸濁液を70μmのフィルターを通し、新鮮な50 mLコニカルチューブに回収し、チューブ当たり15 mlの最終量に調整されます。 </li><li>死んだ細胞や組織の残骸からさらに別の生細胞に、腫瘍細胞懸濁液をFicoll - Paque Plusを使用して密度遠心分離によって分離される。をFicoll - Paqueプラスの下敷きは、プラス徐々に二つの層を混在しないように注意して細胞懸濁液の下をFicoll - Paque 15mLをペッティングすることによって作成されます。 </li><li>ブレーキをオフにして細胞懸濁液とフィコール層は30分間500分の1 gで遠心分離されています。 </li><li>をFicoll - Paqueプラスと細胞解離バッファーの界面で細胞を回収し、新鮮な50 mLコニカルチューブに移しています。 </li><li>新鮮なDMEMに10％FCSを添加することは、それを希釈するために細胞懸濁液に添加される1:3（例：20mLの新鮮な培地は、細胞懸濁液を10mLに追加された）。 </li><li>細胞を10分間、450 gで遠心分離によって収集され、メディアはデカントし、細胞ペレットは、ALDEFLUOR緩衝液1 mLに再懸濁する。 </li><li>細胞懸濁液10μlを10μLトリパンブルーと生細胞血球計数器を使用してカウントしているで希釈する。死んだ細胞や組織の破片が多数このステップ中に明記されている場合は、密度遠心分離による細胞の分離は、（ - 3.7ステップ3.3）を繰り返すことができます。 </li></ol> 4。蛍光活性化細胞選別のための細胞を染色<ol><li>としてラベルseven 12 × 75 mmのポリスチレン試験管は次のとおりです。 <ol><li>汚点のない</li><li> CD44 - APC </li><li> CD24 - PE </li><li>マウスCD31 -ビオチン+マウス系統-ビオチン+マウスH - 2KD -ビオチン</li><li> ALDEFLUOR </li><li> ALDEFLUOR + DEAB + IgG抗体2bκ- APC + IgG抗体2aκ- PE </li><li> ALDEFLUOR + CD44 - APC + CD24 - PE +マウスCD31 -ビオチン+マウス系統-ビオチン+マウスH - 2KD -ビオチン。 </li></ol></li><li>百万円50〜10細胞/ mLの最終濃度にALDEFLUORの緩衝液2 mLの細胞懸濁液を希釈し、氷上に置きます。チューブ＃1、2、3、および4に細胞懸濁液100μLを加え、氷上で保管して下さい。チューブ＃1〜6μLDEABを追加し、氷上に置きます。 1000希釈、残りの細胞懸濁液にALDEFLUOR試薬を追加 - 倍（例えば1.6 mLの細胞懸濁液1.6μLALDEFLUOR試薬を加える）、よく混ぜて、そして氷上に置きます。チューブ＃7に100チューブ＃5と6にこの混合物のμL、および残りの混合液（1.4 mL）を転送する。 40分37℃の水浴中でチューブのすべてをインキュベートする。チューブの底に沈降する細胞を防止するために細胞懸濁液は10分ごとに混ぜる。 </li><li> 450 gでのセルと4を遠心℃で10分間、その後バッファーをデカントするため。 100μLALDEFLUORバッファー中で1位と5管でペレットを再懸濁します。チューブ＃2、3、4、および6に、次のように希釈し、適切な抗体を含むALDEFLUOR緩衝液100μLを追加します。IgG抗体2bΚ- APC、IgG抗体2aΚ- PE、CD44 - APC、およびCD24 - PEの1時20分抗体、マウスCD31 -ビオチン、マウス系統-ビオチン、およびマウスH - 2KD -ビオチン抗体の1:100。チューブ＃7を追加CD44 - APCの1:20希釈を含むALDEFLUORバッファー1.4 mLを加え、CD24 - PE抗体とマウスCD31 -ビオチン、マウス系統-ビオチン、およびマウスH - 2KD -ビオチンの1:100希釈へ抗体。 10分間氷上で細胞懸濁液をインキュベートする。 </li><li> 450 gでのセルと4を遠心℃で10分間、その後バッファーをデカントするため。チューブ＃1、2、3、5、および100μLALDEFLUORバッファ6にペレットを再懸濁します。ストレプトアビジン- PerCP蛋白質の100倍希釈液を含むALDEFLUORバッファーを1.5 mlを準備します。チューブに100μLを追加して＃4とチューブ＃7に1.4 mLを加える。 10分間氷上で細胞懸濁液をインキュベートする。 </li><li> 450 gでのセルと4を遠心℃で10分間、その後バッファーをデカントするため。チューブ＃1、2、3、4、5、および200μLALDEFLUORバッファ6にペレットを再懸濁します。濃度2μg/ mLのヨウ化プロピジウムを含むALDEFLUORバッファー2.8 mLのチューブ＃7でペレットを再懸濁する。すべての回でチューブを氷中に保管してください。 </li><li>ストレーナーのキャップで12 × 75 mmのポリスチレン試験管を用いて35μmのフィルターを介して細胞を渡します。 </li></ol> 5。蛍光活性化細胞選別により、がん幹細胞を分離<ol><li> FACSAriaフローサイトメーターは、細胞選別のために準備されます。 </li><li>補償のコントロールは、チューブ＃1、2、3、4、および5からセルを使用して設定されます。 </li><li>ゲイツは、細胞のシングレットを収集するために前方および側散乱パラメータに基づいて作成されます。 </li><li>非マウス（PerCP陰性）と実行可能な（非ヨウ化プロピジウムで染色）細胞は、FL - 3チャンネルを使用してゲートです。 </li><li> DEAB処理した細胞（チューブ＃6）とFL - 1チャンネルを使用して作成されたALDH +細胞は、ゲートに基づいて収集されます。 </li><li> CD44 + CD24 +細胞は、ゲートに基づいて収集されているFL - 4とFL - 2チャネルを使用してALDEFLUOR、IgG抗体2bΚ- APC、およびIgG2aκ- PE（チューブ＃6）で染色した細胞を使用して作成した。 </li><li>細胞を10％FCSを補充したDMEM 1mLを含む12 × 75 mmのポリスチレン試験管に集められています。 </li><li>細胞がソートされた後、デカント10分、メディアのために450 gで細胞懸濁液を遠心し、500μLの新しいDMEMに懸濁し、10％FCSを補充した。 </li><li>としてステップ3.8節血球計数器を用いて選別された細胞の数を数えます。 </li></ol> 6。代表的な結果このプロトコルは、ALDH +とCD44 + CD24 +膵臓のCSCの分離につながる。マウス由来の細胞の割合は可変ですが、我々は、ほとんどのヒト膵臓癌の異種移植片は、マウスCD31を用いて、マウス由来の細胞、マウスの系統のカクテル、そしてマウスH - 2KDビオチン標識抗体（図1A 20〜60％が含まれていることを見出した）。同様に異なる異種移植片から各CSCの人口の周波数は可変であるが、一般的に我々は、総細胞集団の1-4％がALDH +（図1B）であり、0.2から5パーセントは、CD44 + CD24 +（図1C）であることを見出した。 FACSAriaのマシンの数がしばしば原因FACSAriaによって検出される非細胞性粒子に不正確なので、我々は日常的に手動で血球計数器を使用してソートされた細胞を数える。 <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="図1" /> 図1。膵臓癌の異種移植片からの特定の集団を描いた、フローサイトメトリープロット。（）FL - 3チャネル（ヨウ化プロピジウム+、マウスCD31 +マウスの系統+、またはマウスH - 2KD +）に積極的に染色細胞は分離するように除外されています実行可​​能と非マウス由来の細胞。 （B）ヒト膵臓癌異種移植におけるALDH活性はALDH1の阻害剤ジエチルアミノベンズアルデヒド（DEAB）の存在下および非存在下でALDEFLUOR試薬を用いたフローサイトメトリーにより測定した。フレームは、DEAB処理した細胞に基づいて作成し、未処理の細胞に適用されたALDH +細胞を描くゲートを表しています。 ALDH +細胞の割合がゲートに示されています。 （C）CD44 + CD24 +ゲートは、ALDEFLUORで染色した細胞、およびIgG2bκ- APC（CD44 - APC用アイソタイプコントロール）およびIgG2aκ- PE（CD24 - PEのアイソタイプコントロール）抗体に基づいて作成されました。 CD44 + CD24 +細胞の割合は、ゲートに示されています。

<ol>
	<li>Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells--perspectives+on+current+status+and+future+directions:+AACR+Workshop+on+cancer+stem+cells.">Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells.</a> Cancer Res. 66, 9339-9344 (2006).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 3983-3988 (2003).</li><li>Ginestier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALDH1+Is+a+Marker+of+Normal+and+Malignant+Human+Mammary+Stem+Cells+and+a+Predictor+of+Poor+Clinical+Outcome.">ALDH1 Is a Marker of Normal and Malignant Human Mammary Stem Cells and a Predictor of Poor Clinical Outcome.</a> Cell stem cell. 1, 555-567 (2007).</li><li>Jones, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+clonotypic+B+cells+in+classic+Hodgkin+lymphoma.">Circulating clonotypic B cells in classic Hodgkin lymphoma.</a> Blood. 113, 5920-5926 (2009).</li><li>Matsui, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+multiple+myeloma+progenitors,+stem+cell+properties,+and+drug+resistance.">Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance.</a> Cancer Res. 68, 190-197 (2008).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+pancreatic+cancer+stem+cells.">Identification of pancreatic cancer stem cells.</a> Cancer Res. 67, 1030-1037 (2007).</li><li>Hermann, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+populations+of+cancer+stem+cells+determine+tumor+growth+and+metastatic+activity+in+human+pancreatic+cancer.">Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.</a> Cell stem cell. 1, 313-323 (2007).</li><li>Rasheed, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+tumorigenic+cells+with+mesenchymal+features+in+pancreatic+adenocarcinoma.">Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma.</a> J Natl Cancer Inst. 102, 340-351 (2010).</li><li>Rubio-Viqueira, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+platform+for+translational+drug+development+in+pancreatic+cancer.">An in vivo platform for translational drug development in pancreatic cancer.</a> Clin Cancer Res. 12, 4652-4661 (2006).</li><li>Jones, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+aldehyde+dehydrogenase+in+viable+cells.">Assessment of aldehyde dehydrogenase in viable cells.</a> Blood. 85, 2742-2746 (1995).</li></ol>

利害の衝突は宣言されません。

このプロトコルは、ヒトの異種移植片からの膵臓のCSCの分離を説明します。この手順では、いくつかの重要な手順は、実行可能なCSCがの収量を向上させます。膵臓のCSCの純粋な人口の回復はFACSAriaでの並べ替えの前に細胞懸濁液中に存在する生細胞の純度に依存しています。最大直径1センチ未満の異種移植片を使用するように注意しながら腫瘍の中心部に壊死組織の量を減らすのに役立ちます。さらに、をFicoll - Paque勾配遠心分離時の組織の残骸や死細胞の細胞懸濁液を破壊することは非常に重要ですし、細胞を血球計でカウントされているときに組織の破片や非生存細胞の数が多いが検出された場合に繰り返すことができます。 ここで説明する染色のプロトコルは、特定の細胞表面抗原を検出するためのALDH活性だけでなく、蛍光色素標識抗体を検出するためにALDEFLUOR試薬のシステムを利用しています。 ALDEFLUOR染色は37℃と、それゆえ、37℃で起こることができる、キャッピング抗体の可能性を防ぐために（氷上）抗体染色の前に完了する必要がある℃を実行される細胞がALDEFLUOR試薬を排出できるので、それがALDEFLUORの染色が完了した後4℃でALDEFLUORバッファーで細胞を維持することが重要です。 ALDEFLUORバッファーはALDEFLUORの細胞内レベルを維持できるように薬剤排出抑制剤が含まれています。 このメソッドは実行可能な膵臓CSCを分離するので、それらはこれらの細胞の生理的機能を測定する実験の数に適用することができます。我々は、免疫不全マウスおよびin vitroでの細胞移動/浸潤アッセイを利用した腫瘍イニシエーションアッセイのためにこれらの細胞を使用している。さらに、RNA、DNA、及びタンパク質は、CSCと非CSC集団を比較分析研究のためにこれらの細胞から採取することができる。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM </td>
<td>&nbsp;</td>
<td>Invitrogen (Carlsbad, CA) </td>
<td>11965-118 </td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum </td>
<td>&nbsp;</td>
<td>Harlan (Indianapolis, IN)</td>
<td>BT-9501</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15140-122 </td>
<td>&nbsp;</td>
<tr>
<td>Collagenase type IV </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>17104-019 </td>
<td>&nbsp;</td>
<tr>
<td>Dispase </td>
<td>&nbsp;</td>
<td>Sigma (St. Louis, MO) </td>
<td>D4818 </td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm culture dish </td>
<td>&nbsp;</td>
<td>BD Biosciences (San Jose, CA) </td>
<td>353003 </td>
<td>&nbsp;</td>
<tr>
<td>70-&mu;m filter </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352350 </td>
<td>&nbsp;</td>
<tr>
<td>50-mL conical tube </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352070 </td>
<td>&nbsp;</td>
<tr>
<td>Ficoll-Paque Plus </td>
<td>&nbsp;</td>
<td>GE Healthcare (Upsala, Sweden) </td>
<td>17-1440 </td>
<td>&nbsp;</td>
<tr>
<td>ALDEFLUOR reagent system </td>
<td>&nbsp;</td>
<td>Stem Cell Technologies (Vancouver, BC, Canada) </td>
<td>01700 </td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15250-061 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352054 </td>
<td>&nbsp;</td>
<tr>
<td>CD44-APC (clone G44-26) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>559942 </td>
<td>&nbsp;</td>
<tr>
<td>CD24-PE (clone ML5) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555428 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse CD31-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553371 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse lineage-biotin </td>
<td>&nbsp;</td>
<td>Miltenyi Biotec (Auburn, CA) </td>
<td>130-092-613 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse H-2Kd-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553564 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2b&#x03BA;-APC</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555745 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2a&#x03BA;-PE</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555574 </td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin-PerCP protein </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>554064 </td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4170 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes with strainer caps </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352235 </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of stem cells from human pancreatic cancer xenografts

がん幹細胞（CSC）が悪性腫瘍の増加に同定されており、機能的に自己再生を受け、分化した子孫1を生成する能力によって定義されます。これらのプロパティは、免疫不全マウスに注入するとCSCは、元の腫瘍を再現することができます。上皮悪性腫瘍内のCSCは、最初の乳癌で記述し、特定の細胞表面抗原の発現（CD44 + CD24 低/ - ）を表示することが見出された2。それ以来、CSCは、CD44とCD24を使用して、他のヒト悪性腫瘍の増加だけでなく、他の表面抗原の数で確認されている。アルデヒド脱水素酵素（ALDH）活性、生理的な特性は、、また悪性組織3月5日からCSCを分離するために使用されています。 最近、我々と他の人はALDH活性と細胞表面抗原CD44とCD24、およびCD133 6-8の発現に基づいて、膵臓腺癌からCSCを同定した。これらの高度に腫瘍発生の集団はまたは重複になると他の機能が表示されない場合があります。我々が発見したALDH +とCD44 + CD24 +膵臓のCSCは、同様に腫瘍形成であるが、ALDH +細胞は、比較的多くの侵襲的な8である。このプロトコルでは、継代数の低い人間の異種移植片9から実行可能な膵臓CSCを分離する方法を説明します。異種移植腫瘍は、マウスから採取し、単一細胞懸濁液に作られています。組織の残骸や死細胞は生細胞から分離し、CD44とCD24に対する抗体を使用し、ALDEFLUOR試薬、ALDH 10の蛍光基質を用いて染色される。 CSCは、蛍光活性化細胞選別によって分離されています。隔離されたCSCはその後、生細胞を必要とする分析的または機能的アッセイに使用することができます。

Watch this Scientific Journal Video about Isolation of Stem Cells from Human Pancreatic Cancer Xenografts at JoVE.com

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

がん幹細胞（CSC）が悪性腫瘍の数が同定されている。このプロトコルでは、ヒト膵臓腺癌の異種移植片からCSCを分離するアルデヒド脱水素酵素活性とCD44とCD24の発現を利用したフローサイトメトリー法を説明します。これらの生細胞は、機能的分析の研究で使用することができます。

ヒト膵臓癌異種移植片由来の幹細胞の分離

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

isolation-of-stem-cells-from-human-pancreatic-cancer-xenografts

Research

JoVE Journal

Biology

22.3K ビュー.  Johns Hopkins University School of Medicine. がん幹細胞（CSC）が悪性腫瘍の数が同定されている。このプロトコルでは、ヒト膵臓腺癌の異種移植片からCSCを分離するアルデヒド脱水素酵素活性とCD44とCD24の発現を利用したフローサイトメトリー法を説明します。これらの生細胞は、機能的分析の研究で使用することができます。

ビデオ: Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

プラセンタから造血幹細胞の単離と解析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

生物学

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

人間の膵島単離：パートI：膵組織の消化とコレクション

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

マウスの眼から網膜幹細胞の単離

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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

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

ヒト皮膚から大人上皮幹細胞の単離と培養

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

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 ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

原発腫瘍から免疫細胞の分離

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

人間の脂肪吸引物からの脂肪由来幹細胞を手動で隔離

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...