この作品は、アレクサンダー·フォン·フンボルト財団（5090371）、アレルギーの研究教育のためのクリスティンキューネセンター（CK-CARE）、およびバイエルンStaatsministerium（KDへ）によってサポートされていました。

1。プライマリ線維芽細胞培養からSKPアイソ<ol><li>線維芽細胞培養物のいずれか細胞バンクから、または直接、皮膚生検から得られた15％ウシ胎児血清、2mMグルタミン、10 mg / mlのペニシリン、および10 mg / mlのストレプトマイシンを含有する繊維芽細胞増殖培地DMEMで培養で維持される。 </li></ol>ヒト線維芽細胞GMO3349CとGMO8398Aは、医学研究のための医学研究所（キャムデン、NJ）から得られた結果であり、本研究で使用した。 <ol start="2"><li>集団倍加から培養物（PPDファイル）が20〜35は80％の集密度でSKP培養に用いた。 One 10センチ組織培養ディッシュ（BDファルコン）は、約1.5×10 6個の細胞が含まれています。 </li><li> ℃で37℃で1時間、2ミリリットルトリプシン溶液（0.25％、Invitrogen社）を含むPBSとインキュベートして細胞を洗浄</li><li> 5ミリリットルのPBSでプレートから細胞を回収し、15ミリリットルファルコンチューブに細胞懸濁液を移す。 </li><li> 24時間4℃で細胞をインキュベート。 </li><li> DMEM-F12、3:1（V / V）および40 ngの/ mlのFGF2（BD Biosciences社、20 ngの/ mlのEGF（BD Biosciences社）、B27血清フリーサプリメント2％（Invitrogen）を、1μgの成るSKP増殖培地を調製/ mlのファンギゾン（Invitrogen社）および25μmの/ mlのゲンタマイシン（Invitrogen社）。 </li><li>ペレットを5分間1,200 rpmで細胞および4ミリリットルSKP増殖培地中で直接細胞ペレットを再懸濁し。 25cm 2の組織培養フラスコ（BDファルコン）に細胞懸濁液を移す。 </li><li> 37℃でフラスコをインキュベートすると、3〜4週間毎日球形成のために文化を監視します。 </li></ol> 2。球の培養条件<ol><li> 37°C、5％CO 2で25cm 2のフラスコ（BDファルコン）における培養細胞。 </li><li>フラスコの底に付着した細胞を回避するために、積極的に毎日のフラスコを振る。必要であれば、接着細胞を切り離し、新しいフラスコに文化を転送するには、2 mlの滅菌ピペットでピペッティングと。 </li><li>最初の球は3トン以内の構築を開始O 4日。 </li><li> 3日おきにフラスコの底に球が沈殿しましょう​​と培地の半分を変更してください。成長因子の同一の最終濃度を維持するためには、新たに調製したSKP増殖培地に2X成長因子を追加します。 </li><li> 4ミリリットル最大音量を一定に保つ。 </li><li>球が〜200mmのサイズに達すると、それらは激しく2ミリリットルピペットで上下にピペッティングによって小さいサイズに球を分解することによって継代されるべきである。 </li><li>文化は）ステップ2.4で説明したようにフィード二つ25cm 2のフラスコ（BDファルコン）と球培養に分割されます。 </li><li>球体21日16によってスクリーニング幹細胞マーカーのために収集される。 </li></ol> 2.6から手順）と2.7）が（1）〜球の伝播を記述する各球内のすべてのセルにアクセスするために、（2）先使用をSKP球文化を拡大するSKP培地に含まれる栄養分や成長因子を可能にします。 当社CU下通常スフィア培養lture条件は、3ヶ月の期間を上に成長する能力を維持し、3〜4回の間で継代する。 3。幹細胞マーカーのための免疫細胞化学<ol><li>球培養物を、16日目で21にPBSで収穫される。文化は5分間1,000 rpmでペレット化されています。 </li><li>球はPBSを少量に再懸濁する。 </li><li>顕微鏡スライド（フィッシャーブランド）で直径約0.5μmのDAKOペンで二つの円を描画します。 </li><li>サークルに球懸濁液50μLを加える。 </li><li>各ドロップ球の存在を光学顕微鏡で確認してください。 </li><li>ボンネットの下に滴が乾燥してみましょう。 </li><li>どちら-80℃でスライドを凍結または免疫蛍光染色プロトコルを進める。 </li><li>免疫蛍光染色のために、10分間-20℃で予冷メタノール（100％）でスライドを固定します。 </li><li> PBSでスライドを洗浄します。 </li><li> PBS/10％ウシ胎児血清/ 0.02％トリトンX100でスライドをブロック少なくとも1時間。 </li><li> PBS/10％4で室温で2時間または一晩のためにFBS℃まで：ブロッキングバッファーで希釈した一次抗体（ 例えば 、抗ネスチン、抗Oct4の、抗TG30、抗Nanogの抗体を、 表3）とインキュベート</li><li>ブロッキング緩衝液で3回洗浄し、室温で1時間二次抗体と共にインキュベートする。 </li><li> PBSでバッファと3回をブロックで3回洗浄します。 </li><li>ベクタシールドマウント培地（株式会社ベクター）でスライドをマウントします。 </li><li>免疫蛍光顕微鏡検査によって幹細胞マーカーの発現のためのサンプルを分析する。 </li></ol> 4。幹細胞マーカーの発現レベルのリアルタイムPCR解析<ol><li> 18日目から21日まで約2〜3ミリリットルのスフィアの文化を取る。 </li><li>ペレットを5分間1,200 rpmで球とすべて培地を吸引。 </li><li>をRNeasyミニキット（キアゲン、バレンシア、CA）を使用して、球からRNAを分離します。 </li><li>分析し、アガロースゲルでのRNA純度を評価する。&lt;/李&gt; <li>テンプレートとして全細胞RNAを用いて（オムニスクリプト逆転写酵素（キアゲン））cDNAを合成。 </li><li> 20μlの各プライマーの375 nmおよびテンプレート50ngの濃度のパワーのSYBR Green PCRマスターミックス（Applied Biosystems社）における幹細胞マーカー（ 表1に示されている）のためのリアルタイム-PCRプライマーを用いによって幹細胞マーカーを検証反応容積。 GAPDHは、内在性コントロールとして使用される。 </li><li>増幅のために95℃で初期変性95℃で40サイクル℃で5秒、20秒のために60°CのためのCに続いて2.5分のためのCを使用しています。 </li><li> 2（ΔΔCT）法8と走りを分析します。 </li></ol> 5。平滑筋細胞内に向け分化<ol><li>一日21〜26球はSKP培地中で6ウェル培養皿（BDファルコン）に播種した。 </li><li>細胞は、72時間付着し、SKP培地中球から脱却させた。 </li><li>平滑筋細胞（平滑筋細胞）differentiatイオンは、高グルコース5％FBS、5 ngの/ mlのPDGF-BB（Invitrogen社）、および2.5 ng / mlのTGF-B1（Invitrogen）を含むダ​​ルベッコ改変イーグル培地（Invitrogen）を成るSMC分化培地で培地に交換することによって開始開始。 </li><li>媒体は文化で、3〜4週間の期間のために新たに調製SMC分化培地で3日ごとに変更されました。 </li><li> SMCマーカーのスクリーニングは、免疫組織化学によって、3〜4週間後に行った。 </li><li>細胞を10分間、PBS中の4％パラホルムアルデヒド溶液で固定した。 </li><li>細胞をPBSで30分間、0.3％トリトンX-100を含有する透過処理した。 </li><li>細胞は、3.15へ）を室温で1時間αSMA（MO851、ダコ、1:100）、またはカルポニン（M3556、ダコ、1:100）に対する抗体でインキュベートし、3.12で説明したようにさらに処理された）。 </li></ol>

<ol>
	<li>Jahoda, C. A., Whitehouse, J., Reynolds, A. J., Hole, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+dermal+cells+differentiate+into+adipogenic+and+osteogenic+lineages.">Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages.</a> Exp. Dermatol. 12, 849 (2003).</li><li>Watt, F. M., Celso, L. o., C, V., Silva-Vargas, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+stem+cells:+an+update.">Epidermal stem cells: an update.</a> Curr. Opin. Genet. Dev. 16, 518 (2006).</li><li>Blanpain, C., Horsley, V., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+stem+cells:+turning+over+new+leaves.">Epithelial stem cells: turning over new leaves.</a> Cell. 128, 445 (2007).</li><li>Hunt, D. P., Jahoda, C., Chandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+skin-derived+precursors:+from+biology+to+clinical+translation.">Multipotent skin-derived precursors: from biology to clinical translation.</a> Vurr. Opin. Biotechnol. 20, 522 (2009).</li><li>Toma, J. G., et al. <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+multipotent+adult+stem+cells+from+the+dermis+of+mammalian+skin.">Isolation of multipotent adult stem cells from the dermis of mammalian skin.</a> Nat. Cell Biol. 3, 778 (2001).</li><li>Fernandes, K. J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dermal+niche+for+multipotent+adult+skin-derived+precursor+cells.">A dermal niche for multipotent adult skin-derived precursor cells.</a> Nat. Cell Biol. 6, 1082 (2004).</li><li>Wenzel, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na&#239;ve+adult+stem+cells+from+patients+with+Hutchinson-Gilford+progeria+syndrome+express+low+levels+of+progerin+in+vivo.">Na&#239;ve adult stem cells from patients with Hutchinson-Gilford progeria syndrome express low levels of progerin in vivo.</a> Bio. Open. 1, (2012).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25, 402 (2001).</li><li>Biernaskie, J. A., McKenzie, I. A., Toma, J. T., Miller, F. D. <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+skin-derived+precursors+(SKPs)+and+differentiation+and+enrichment+of+their+Schwann+cell+progeny.">Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny.</a> Nature Protocol. 1, 2803 (2006).</li><li>Toma, J. G., McKenzie, I., Bagli, D., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+multipotent+skin-derived+precursors+from+human+skin.">Isolation and characterization of multipotent skin-derived precursors from human skin.</a> Stem Cells. 23, 727 (2005).</li><li>Fernandes, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+neurogenic+potential+of+multipotent+skin-derived+precursors.">Analysis of the neurogenic potential of multipotent skin-derived precursors.</a> Electrophoresis. 201, 32 (2006).</li><li>Hill, l. K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+vascular+progenitor+cells+capable+of+endothelial+and+smooth+muscle+cell+function.">Human embryonic stem cell-derived vascular progenitor cells capable of endothelial and smooth muscle cell function.</a> Exp. Hematol. 38, 246 (2010).</li></ol>

我々は、開示することは何もありません。

本明細書に記載した方法を用いて、ナイーブ皮膚幹細胞は、一次皮膚線維芽細胞培養物から単離することができる。このアプローチを使用して、我々は最近まれな遺伝症候群、ハッチンソン·ギルフォード早老症候群7患者由来線維芽細胞培養からの成体幹細胞の単離と特性を報告した。としてそれらの前駆細胞を発現幹細胞マーカーは自己再生が可能であり、（お願いヴェンツェル<e...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Invitrogen</td>
			<td>31966-047</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM low glucose</td>
			<td>Invitrogen</td>
			<td>21885-108</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>10270-106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td>Final conc.: 200 mM</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Final conc.: 10 mg/ml /10 mg /ml</td>
		</tr>
		<tr>
			<td>trypsin solution (0.25%)</td>
			<td>Invitrogen</td>
			<td>25200-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Nutrient Mixture (Ham)</td>
			<td>Invitrogen</td>
			<td>21765-029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>BD Biosciences</td>
			<td>4114-TC-01M</td>
			<td>Final conc.: 40 ng/ml</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>BD Biosciences</td>
			<td>236-EG-200</td>
			<td>Final conc.: 20 ng/ml</td>
		</tr>
		<tr>
			<td>PDGFBB</td>
			<td>Invitrogen</td>
			<td>PHG0043</td>
			<td>Final conc.: 5 ng/ml</td>
		</tr>
		<tr>
			<td>TGF-b1</td>
			<td>Invitrogen</td>
			<td>PHG9204</td>
			<td>Final conc.: 2.5 ng/ml</td>
		</tr>
		<tr>
			<td>25 cm2 flask</td>
			<td>Omnilab</td>
			<td>FALC353109</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS w/o CaMg</td>
			<td>Invitrogen</td>
			<td>14190-169</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Roth</td>
			<td>8388.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector Inc.</td>
			<td>H-1200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNeasy Minikit</td>
			<td>Qiagen, Valencia, CA</td>
			<td>74104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Omniscript Reverse Transcriptase</td>
			<td>Qiagen</td>
			<td>205113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SsoFast EvaGreen Supermix</td>
			<td>BioRad</td>
			<td>172-5201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290-018</td>
			<td>Final conc.:1 mg/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Specific reagents and equipment.</td>
		</tr>
	</tbody>
</table>

na&iuml;ve adult stem cells isolation from primary human fibroblast cultures

過去10年間で、いくつかの成体幹細胞集団はヒトの皮膚1-4に同定されている。多能成人皮膚前駆体の単離は、最初の6ミラーF. D実験室5、により報告された。これらの初期の研究では成体哺乳類の真皮5から多能性前駆細胞集団を記述した。これらの細胞は-と呼ばSKPs、皮膚由来の前駆体のためには、 -単離され、げっ歯類とヒトの皮膚から拡大し、そのようなニューロン5などの皮膚に見たことがない種類の細胞を含む神経と中胚葉の両方の子孫へと分化させた。培養SKPs上の免疫細胞化学的研究は、細胞は、フィブロネクチンおよび多能性幹細胞マーカー6に加えて、ビメンチンおよびネスチン、神経および骨格筋において発現前駆体中間体フィラメントタンパク質を発現することを明らかにした。今までは、成体幹細胞集団のSKPsを、新たに収集された哺乳動物の皮膚生検から単離されている。 ve_content &quot;&gt;最近、我々は皮膚の人口に由来前駆細胞は、皮膚生検7から確立された主要な線維芽細胞培養中に存在し続けたことを確立し、報告している。少数体性幹細胞は、早期の集団倍加一次線維芽細胞培養にある可能性があることは前提だった（1）SKPs及び一次線維芽細胞培養は、真皮から誘導されるので、SKP少数の細胞は、一次皮膚線維芽細胞培養物とされている凍結アリコートから成長した（2）一次線維芽細胞培養物中に存在残る可能性が以下の観測結果に基づいて7生残った少数の細胞を含有する貯蔵又は転送時に好ましくない温度にさらさこれらの稀な細胞を数回拡大すると、継代され得ることができた。この観察は示唆高い増殖能やストレスに対する抵抗性を有する細胞の数が少ないヒト線維芽細胞培養7に存在していた。我々は、世界中の組織バンク（ 図1）から容易に入手可能である一次線維芽細胞培養からの成体幹細胞の迅速な分離のためのプロトコルを確立するために、これらの知見を利用した。線維芽細胞培養がで稀な遺伝性疾患と同様にモデリング疾患の根底にある分子機構を分析するための新しい機会を開くため、組織バンクから入手可能かもしれないが皮膚サンプルにアクセスできないときには前駆細胞の単離を可能にするため、この方法では、重要な意義を持っているディッシュ。

我々は、最近7を報告したように選択的にEGFとFGF2からなる制御された成長条件の下でSKP球を生成するために展開した細胞の集団は、一次皮膚線維芽細胞培養（ 図1）中に存在することを示している。 典型的には、細胞バンクから入手可能な一次線維芽細胞株に対応するPPDは15〜25線維芽細胞培養は、本研究で使用した。この方法で説明した24時間のため...

Watch this Scientific Journal Video about NaÃ¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures at JoVE.com

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

私たちは、初代ヒト線維芽細胞培養からナイーブ多能皮膚由来の前駆体（SKP）細胞を単離する方法を報告する。我々は、線維芽細胞培養物に由来するこれらのSKPsは、ヒト皮膚生検から直接誘導されるものに類似した幹細胞の特性を共有することを示している。これらの細胞は、そのようなOCT4とNanogのような多能性マーカーに加えて、神経堤マーカー、ネスチンを発現。

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 ...

nave-adult-stem-cells-isolation-from-primary-human-fibroblast-cultures

Research

JoVE Journal

Biology

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

14.1K ビュー.  Technische Universität München. 私たちは、初代ヒト線維芽細胞培養からナイーブ多能皮膚由来の前駆体（SKP）細胞を単離する方法を報告する。我々は、線維芽細胞培養物に由来するこれらのSKPsは、ヒト皮膚生検から直接誘導されるものに類似した幹細胞の特性を共有することを示している。これらの細胞は、そのようなOCT4とNanogのような多能性マーカーに加えて、神経堤マーカー、ネ?...

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

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 ...

生物学

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 ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

外植片から成長した初代ヒト気管支上皮細胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

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

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

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

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

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

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

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

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

培養ヒト胚および誘導多能性幹細胞に適したマウス胚性線維芽細胞の調製

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

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

高密度ヒト胚性幹細胞の培養線維芽細胞からの迅速な除去

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

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 ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

マウスおよびヒト結腸組織からの一次筋線維芽細胞の分離

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...