著者は、現在のフィルムとスライドの編集上の彼らの助けのための氏カイルホールデン氏とジョナサンルーカスのための細胞分離に与える技術的なヘルプのために氏鷹パンとイアンBellayrの大きく感謝しています。博士：過​​去数年にわたって修正されたpreplate技術の開発に関わってきた人の、以下の一覧に感謝。 Burhan Gharaibeh、宝洪曹、Deasyブリジット、トーマスR.ペイン、愛平陸、海融鵬、秀樹大島、ボー曽、暁東ムー、Kimimasa飛田、ロンJankowski、Makato池澤。我々は、James H.カミンズ、ライアンPruchnic、ジョセフFeduska、レベッカC. Schugar、海鷹パンとジェシカTebbetsからの技術支援に感謝しています。著者はまた、筋ジストロフィー協会（MDA）、国立衛生研究所（NIH）、国防総省（DOD）、UPMCのピッツバーグの小児病院、科からこの仕事のための財政支援に感謝整形外科、およびピッツバーグ大学。

一般的には、完全な修正preplateの手順は、準備の1〜2日、技術的な手順を実行するの1週間、免疫細胞と細胞の同定の2〜3日、および幹細胞集団の拡大のさらに2〜3週間を必要とします。幹細胞培養に必要な機器は、ベンチトップ遠心機、CO 2インキュベーター、層流の組織培養フードと蛍光とデジタルカメラを搭載した倒立顕微鏡を含む他の細胞培養システムのそれに似ています。分離された幹細胞は、クローニングすることができると、現在または将来の研究の1,2のための液体窒素中で長期的に保存することができます。この手順で使用されるすべての試薬および材料は滅菌と無菌的に処理する必要があります。 ステップ1：準備<ol><li>コラーゲンコートの組織培養ディッシュやフラスコ：子牛の皮（1リットルで0.1グラム、Sigma - Aldrich社）から派生したI型コラーゲン。コー​​トへのプラスチック容器は、次のとおりとして1,2の前に説明したT​​ - 25フラスコ、6または12ウェルプレート、皿を（60、100ミリメートル、BDファルコン）。ゆっくりと4時間のコラーゲンにもコーティングを確保するために、プラスチック容器を30分おきに振る。コラーゲン溶液は、その後削除されます。とコーティングされたプラスチック製品は、無菌性を確保するために、UV光とフードとの両方の組織培養フード内で乾燥させる、そして最終的にrecappedまたは後で使用するためにカバー。 </li><li> 、、50mLの熱不活性化ウシ胎児血清（FBS、インビトロジェン社）、50mLのウマ血清（HS、Invitrogen社）、及び5mLのニワトリ胚抽出物（CEE、正確な化学株式会社400ML DMEM（Invitrogen社DMEM、高グルコース）：細胞培養​​の培地を準備）使い捨て0.22μmの500mlの滅菌フィルターシステム（コーニング）を使用してフィルタ処理された真空になります。無菌ペニシリン/ストレプトマイシン溶液（Invitrogen）を最終的に100単位/ mLを追加されます。調製された溶液は、少なくとも一ヶ月に4℃で保存することができます。このソリューションは、ときに文化を分割または移植用細胞を調製フラスコから細胞を剥離するために使用されます。 </li><li>細胞分離のためのソリューションをウォームアップ：下記の試薬を37にウォームアップする必要があります° C細胞分離のためのその使用前に：ハンクのバッファ塩溶液（HBSS、Invitrogen社）、0.2％（wt / vol）のコラゲナーゼタイプXI（シグマmLのHBSS 3,200コラーゲン消化単位の平均で- Aldrich社）、;ディスパーゼ2.4 units/1mL（Invitrogen社製）、0.5％（wt / vol）のトリプシン（インビトロジェン）。 </li><li>準備のために外科手術機器の滅菌：異なるサイズの鉗子、マイクロはさみ、アイリスはさみおよび/または外科用メス刃：手術の手順は、含む滅菌手術器具が必要です。また、15および50 mLのポリプロピレン遠心チューブ（BDファルコン）、セルストレーナー（孔径70メートル）（BDファルコン）、18 - 、23 - および27ゲージの針（BD PrecisionGlide）、及び10 - または20 - mlの滅菌シリンジ（BD）。 </li></ol>ステップ2：組織生検、単離および機械的解離1月4日 組織生検は無菌培養のボンネットの下に行われ、新鮮な収穫腱や骨格筋を含む、野生型マウスの後肢（女性C57BL/6J、4-5週齢または若い、ジャクソンの脛骨またはヒラメ筋から得られる研究所）。目に見える結合組織、血管、脂肪組織は、生検サンプルから削除されます。腱と筋肉の組織は、慎重に皮膚と骨の残骸を除去する外科解剖顕微鏡下で同定し、含有する冷（4℃）HBSS（FBSの5％を追加で補充）。を皿に置かれている分離された組織は、その後別々に冷HBSSを含むコラーゲンコートディッシュ上に配置されており、マイクロシザー構造および/ ​​またはメス刃を用いて粗いスラリーに（&lt;1x1のmm 3の ）ミンチされます。 ステップ3：組織の酵素消化： みじん切りの組織は、手順2,4,5の一連の後、酵素的に解離している<ol><li>別の15 mlチューブと4時〜3500rpmで遠心° Cで5分にミンチ組織のスラリーを転送する。 </li><li>上清を除去、HBSSで洗浄し、再度遠心操作を繰り返します。 </li><li>上清を除き、あらかじめ温めておいた0.2パーセントコラゲナーゼタイプXI（シグマ）10 mlを加えることによってこれらのスラリーを消化。継続的に穏やかにチューブを揺らしながら、37℃で60分間インキュベートする。また、手で10分ごとに振る。 </li><li> 10ミリリットルディスパーゼ（2.4単位/ mL、Gibco社）溶液中でこれらのスラリーを遠心し、懸濁します。手で振ったり、軽く10分ごとに揺らし、37℃で45分間インキュベートする。 </li><li>遠心分離し、これらのスラリーは、0.2％トリプシンHBSS溶液10mlに再懸濁する。インキュベーションは、単一の細胞を顕微鏡下でサスペンションを観察することによって実行できる組織から放出される範囲に基づいて変化します。それは、反転管で37℃で30分を超えないようにCを推奨したり、静かに10分ごとに揺動されていません。 </li><li>得られた細胞を遠心sと再サスペンド増殖培地（PM）の10mlに細胞ペレットを。 </li><li>針の一連の抽出液を渡すことによって、細胞懸濁液を取り除きます。細胞は、その後、18Gを通して23Gを2回通過し、最終的に27G針されています。 </li><li> 70μmのセルストレーナーを介して細胞抽出液を渡します。 </li></ol>ステップ4：細胞接着率1-3に基づいて異なる細胞集団を分離するPreplating <ol><li>遠心分離し増殖培地で細胞ペレットを再懸濁します。 </li><li>コラーゲンコートT - 25フラスコ（または60 mmディッシュ）上にプレート細胞の混合物とPP1としてマークします。細胞懸濁液を顕微鏡で屈折原子核と他の破片の多数を所有していると観察する必要があります。 </li><li> 37℃で2時間加湿、5％CO 2インキュベーターでC。 </li><li> T - 25フラスコ（または60 mmディッシュ）とマークがPP2となる新しいコラーゲン被覆に非接着細胞を移す。 PP1というラベルの付いたプレートにPMを5mlを追加。フラスコに付着初期の付着した細胞は、主に筋線維芽細胞3です。 </li><li>別の24時間インキュベーターにフラスコを返す、新しいコラーゲンをコートしたT - 25フラスコ（または皿）とPP3としてそれをマークするために非接着細胞を移す。 PP2ラベルの付いたプレートに増殖培地5mlを追加。このフラスコに付着これらの接着した細胞は、主に線維芽細胞2,3です。 </li><li>インキュベーターにフラスコを返し、人口PP6が作成されるまで、別の24時間以内に手順を繰り返します。浮遊細胞の各グループを維持し、それらが増殖し、倒立顕微鏡を使用して定期的にそれらを検査することができます。 PP3 - PP4筋衛星細胞がしばしばPP5 2で主に見ている間にほとんどが筋芽細胞です。 </li><li>徐々に接着した細胞のほとんどは、通常、PP6で取り付けます。この段階で、細胞は小さく、円形、光屈折率を表示され、数は非常に疎であり、幹細胞集団の1,2と見なされます。 </li></ol>ステップ5：隔離された幹細胞の同定と拡大<ol><li>孤立PP6細胞の数が非常に少ないですし、毎日変更されるFBS豊富な増殖培地（DMEM 20％FBS）で維持する必要があります。細胞のほとんどは、培養の次の1-2週間の間にPP6文化のダイに存在しているが、大規模な、健康なPP6人口は、通常、拡張の追加の1-2週間後に作成されます。マウスから単離した幹細胞を高発現幹細胞抗原1（SCA1）、CD34、胎児肝キナーゼ1（Flk1）、および免疫細胞化学染色を介して可視化できる他の幹マーカー1,2,6,7。これらの幹細胞はまた、複数の分化能力3,6,8を示 ​​す。 </li><li>単離された細胞は、分化の1,2を避けるために、培養中の低細胞密度（50〜100細胞/ウェルまたはフラスコやディッシュの&lt;20-30％コンフルエントで12ウェルディッシュ）に維持して展開されます。非常に少数の細胞が膨張中にクローンを形成し、これらのクローンES細胞は、他の能力の研究のための長い時間の増殖（LTP）を受けることができる。クローンES細胞はまた、10％DMSOとPMの培地を、例えば一般的な細胞の保管手順を使用して液体窒素中で保管す​​ることができます。他のバックアップ研究のための幹細胞の低継代株の数を作る。 </li></ol>

<ol>
	<li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu-Petersen, Z. <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+a+novel+population+of+muscle+stem+cells+in+mice:+potential+for+muscle+regeneration.">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li><li>Li, Y. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle.">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li><li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &. a. m. p. ;. a. m. p., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse.">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li><li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics.">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing.">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers.">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential.">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li><li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+based+on+muscle+stem+cells.">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li><li>Gates, C. B., Karthikeyan, T., Fu, F. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells.">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li></ol>

利害の衝突は宣言されません。

それはいくつかの成人組織では、複数の幹細胞が含まれていることが認識されている。研究の過去数年間、幹細胞は骨格筋と腱を含む柔らかい筋骨格系組織で検出されている。成功した腱や骨格筋から成体幹細胞を分離するために我々の研究室で使用されている修正preplateのテクニックとして知られているこの現在のプロトコルは、詳細手順を、。細胞上記の変更preplateのテクニックを使用することをコラーゲンコートしたフラスコ（図1）への接着特性に基づいていくつかの集団に分けることができます。組織内に存在する線維芽細胞および筋線維芽細胞はすぐに24時間から48時間以内にコラーゲンコートしたフラスコに付着し、最初はPP1 - PP2で他の細胞から分離されています。筋原性または内皮細胞は、48〜96時間内でより長い期間、（PP3 - PP5）の後にコラーゲンコートしたフラスコに付着し、収穫し、それらの細胞表面マーカーによって識別することができます。後でpreplated細胞、96時間以降（PP6）は、成体幹細胞（図2）とみなされます。後半preplate細胞は、小さな丸い、およびそれらの分離の初めに半透明表示され、さらに幹細胞の抗原1（SCA1）、CD34、胎児肝臓キナーゼ1（Flk1）の発現のためのフローサイトメトリーまたは免疫細胞化学によって特徴付けることができる、と他の幹細胞マーカー（図3）。中胚葉、外胚葉、および内胚葉9：三胚葉への分化を通じて多能を実証している孤立した幹細胞は自己複製および実験的研究のための能力を持っている。複数の調査は、これらの幹細胞は骨細胞、脂肪細胞、軟骨細胞、および造血細胞6,8を含む中胚葉系列の細胞に分化することが明らかになっている。他の研究では、成体幹細胞は神経細胞およびグリア細胞のマーカーと末梢神経が10を損傷しているafter手足の機能を向上させる能力の彼らの発現を介して外胚葉の細胞系譜に分化することを明らかにした。これらの分離された成体幹細胞が負傷したと病気の筋骨格系の組織を修復するための有益され、in vivo試験に関連する他の治療に心臓、肝臓、脊髄だけでなく、小腸粘膜下組織や膀胱のような特定の医学的条件を含む他の組織で行われているいる腹圧性尿失禁9。 <img src="/files/ftp_upload/2011/2011fig1.jpg" alt="図1" /> 図1。 <img src="/files/ftp_upload/2011/2011fig2.jpg" alt="図2" /> 図2。 <img src="/files/ftp_upload/2011/2011fig3.jpg" alt="図3" /> 図3。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#11995-073</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#10437-028</td>
<td>&nbsp;</td>
<tr>
<td>Heat-inactivated horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#26050-088</td>
<td>&nbsp;</td>
<tr>
<td>Chick embryo extract</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>#CE650T-10</td>
<td>&nbsp;</td>
<tr>
<td>100U ml penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#14190-250</td>
<td>&nbsp;</td>
<tr>
<td>Hank&#x2019;s Buffered Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#24020-117</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase type XI 0.2%(wt/vol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C7657</td>
<td>&nbsp;</td>
<tr>
<td>Dispase2.4U ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA0.5%(wt/vol) (10x;5g liter trypsin (1:250)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15400-054</td>
<td>&nbsp;</td>
<tr>
<td>Collagen for coating flask : Type I collagen derived from calf skin 0.1 g liter</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C-9791</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>#D-2650</td>
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isolating stem cells from soft musculoskeletal tissues

成体幹細胞は、長い再生医療への応用に関して議論されている。成体幹細胞は、多系統の細胞の分化と自己複製能を受けることにより、その印象的な機能に負傷し、病変組織を治療するための興奮を大いに生成している。最も重要なことは、これらの資質は、自家細胞移植療法での使用に有利にしている。現在のプロトコルは、1である筋骨格系の軟部組織、例えば、腱や筋肉、修正されたpreplateのテクニックを読者に紹介する<sup&gt; ST</sup&gt;酵素的に解離し、培地でコラーゲンコーティングしたフラスコ内に配置。培地と、残りの浮遊細胞で構成されている上清を、順次新しいフラスコに毎日転送されます。幹細胞は通常、（シリアル転送またはpreplates）5-7日間かかるているフラスコに付着する最も遅いです。このテクニックを使用することにより、これらの組織中に存在する成体幹細胞は、簡単にかなりの非侵襲的な手続きを経て収穫することができる。

Watch this Scientific Journal Video about Isolating Stem Cells from Soft Musculoskeletal Tissues at JoVE.com

Isolating Stem Cells from Soft Musculoskeletal Tissues

フラスコに細胞の付着の速度に基づいて、筋骨格系の軟部組織から成体幹細胞を単離する。

ソフト筋骨格組織から幹細胞を単離する

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

isolating-stem-cells-from-soft-musculoskeletal-tissues

Nanyang Technological University

Research

JoVE Journal

Biology

13.3K ビュー.  Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC. フラスコに細胞の付着の速度に基づいて、筋骨格系の軟部組織から成体幹細胞を単離する。

ビデオ: Isolating Stem Cells from Soft Musculoskeletal Tissues

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

胚盤胞からマウス栄養膜幹細胞の導出

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

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 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 ...

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 ...

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

マウスの心臓から初代メラノサイト様細胞を単離する

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...