著者は優秀な技術支援のためにpHPLとマルガレッタFrühwirthとダニエラターラーを提供するための博士カタリーナSchallmoserに感謝。

A.）ECFC ISOLATON 1日目： <ol><li>あなたが実験を始める前に細胞培養培地内皮増殖培地-2（EGM - 2）を準備する。 、ヘパリン（1000 U / mlの1ミリリットル）、ペニシリン/ストレプトマイシン（すべてSigma社、セントルイス、MO 5mlの100倍液で500ミリリットル内皮基礎培地- 2を補足<a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> 、L -グルタミン（2mMの最終濃度） ）、および50mlプールされたヒト血小板溶解液（pHPL）。 、0.2 mlのヒドロコルチゾン、2ミリリットルhFGF - B、0.5ミリリットルVEGF、0.5ミリリットルEGF、0.5ミリリットルIGF - 1と0.5ミリリットルアスコルビン酸（SingleQuotsとして提供される、すべてのロンザ、ウォーカー、MDを追加します。 <a href="http://www.lonzabioscience.com/">http://www.lonzabioscience。 COM /</a> ）。無菌ろ過0.2メートル、細孔径500ミリリットルミリポア真空フィルタを通して培地。 pHPLは小さな血栓を形成するので、1つの媒体のための二つのフィルタが必要な場合があります。プリウォームフィルタリングEGM - 2〜37℃の水浴で</li><li> 6ミリリットル防腐剤フリーナトリウムヘパリンの血液バキュテイナ管に肘静脈からのヒト成人末梢血5mlを描画することにより、血液サンプルを収集する。次の2つの時間の最大値内で血液サンプルを処理する必要があります。 </li><li>ベントキャップ、一つ25mlのピペットと層流のベンチで2つ5ミリリットルピペットとフラスコ、一つ75センチメートル2（コスターT75）を準備することから始めます。 </li><li>プレフィル15ミリリットルは、予め温めておいた25mlのピペットとフラスコにEGM - 2を補った。今血液バイアルおよびサンプルフラスコに5 mlのピ​​ペットを用いてすべての5 mlを開きます。目の5 mlのピ​​ペットを用いて追加のEGM - 2の5mlで空の血液バイアルを洗浄します。 T75内の総容積は25 mlです。 37℃インキュベーターにフラスコと場所の通気キャップを閉じ° C、5％CO 2および24時間で95％の湿度。 </li></ol> 2日目： <ol><li>約24時間（一晩）した後は、非接着細胞を取り除くために中期の血液の混合を削除する必要があります。層流でこの準備two 25ミリリットルピペット三5ミリリットルピペット用、プレ暖かい20ミリリットル° Cの水浴中で37にEGM - 2（前日に準備）、30 mlのPBSを補った。 </li><li>インキュベーターからサンプルでフラスコを取り外し、25 mlのピ​​ペットで上清を取り除く。潜在的なコロニーの損傷を防ぐために、フラスコの表面を傷付けないように注意してください。静かに予め温めておいたPBSと傾く一方から他方へのフラスコ10 mlを加えることによって内側のフラスコの細胞接着表面を3回洗浄する。 PBS直ちに毎回と廃棄を削除します。 </li><li>フラスコに新鮮予め温めておいたEGM - 2の20 mlを加え、インキュベーターに戻すに置きます。このプロセスはデ​​タッチしないように接続されたセルに対して可能​​な限り迅速かつ穏やかとして実行する必要があります。彼らは長い間にPBSでまたは室温で保存されている場合ECFCsは容易に切り離す。 </li><li> ECFC伸長を探している光学顕微鏡の低倍率を使用して2日目から始まる全身フラスコ隔日を通じて画面。コロニーは、コロニーの位置を示すフラスコの外側に上にマークを発見された場合。 flaksが5分以上インキュベーターの外であってはならないことに注意してください。 </li></ol> 5日目： <ol><li>五日目に播種した後、非接着細胞を除去する完全培地の変更を行います。 EGM - 2に37℃の水浴中と2つの25 mlのピ​​ペットを準備のこの目的のため事前に暖かい20ミリリットル用。 </li><li> 25mlのピペットを使用して完全培地を取り出して、できるだけ早く、新鮮な、予め温めておいた補充EGM - 2を追加します。細胞が乾燥しないに1分以上のための媒体なしのままにしないでください。インキュベーターに戻して置いて、毎日のフラスコをスクリーニング繰り返す。 </li><li>新鮮な補充EGM - 2で、週に2回培地の30％を置き換えることにより、細胞を養う。 </li></ol> DAY 14から28： <ol><li>石畳の形態を持つ典型的なコロニーは、12日目の周りに表示されます。各コロニーの位置を示すために、フラスコの上部外側にマークを付けます。単一細胞がデタッチを開始しない限り、コロニーは、28日目までに成長することができます。一度、一次コロニーは、それらがその大きさに応じて、14日目と28の間に収穫することができる確認されている。 </li></ol> B.）ECFC HARVEST <ol><li>事前に暖かい5ミリリットルトリプシン0.05％/ EDTA、20 mlのPBSと5ミリリットル新鮮な補充EGM - 2。 </li><li>完全にファルコンチューブ（潜在的なコロニーの損傷を防ぐために、フラスコの表面に触れないように注意を取る）にフラスコから培地を除去し、PBS予熱した10mlで慎重に細胞を洗浄。 </li><li>インキュベーター内で5分間5ミリリットルトリプシン/ EDTAとインキュベートする。それは有毒になるので細胞が以上の7分間トリプシンにさらされるべきではない。徹底的にフラスコの側面をタップすると細胞がデタッチするのに役立ちます。保存され使用される培地の約10mlを加えることによってトリプシンのプロテアーゼ活性を消失。 </li><li> 50ミリリットルファルコに細胞懸濁液を取り除くn個のチューブ。その後収穫された細胞懸濁液に添加されている10 mlのPBSで一回フラスコを洗ってください。 4℃で7分間300gで細胞懸濁液を遠心° Cは、細胞を回収し、トリプシンを除去する。上清を捨て、1mlのPBSですぐに細胞ペレットを再懸濁します。氷上に置きます。 </li><li> R.リカルドとK. PhelaniでJoveのプロトコルで説明されているように細胞数を決定します。5 </li></ol> C.）ECFC EXPANSION <ol><li> 1.1で説明したように）500ミリリットルEGM - 2を補足するものです。 37〜プレ暖かい℃の水浴でC。 </li><li> Nunc社、シカゴ、イリノイ;約2500 cm 2と文化空間を11 T220（225 cm 2）のまたは15 T175（175cm 2の ）またはいずれか4層の細胞の工場を（CF - 4準備<a href="http://www.nuncbrand.com/">http://www.nuncbrand 。com</a> ）。さらに、2つの新鮮なベントキャップと層流ベンチで特別な滅​​菌漏斗が必要になります。 </li><li> 100の開始細胞密度でシードECFCsに細胞数のC / cm 2と計算が必要です。一つはCF - 4 252800細胞を500 mlの新鮮なEGM - 2に再懸濁させ、滅菌漏斗を使用してCF - 4に注いだ。 Closeをベントキャップ付き通気口とすべての4小麦粉との間の媒質の平等な分配を可能にする、CF - 4対1の経度の側に傾けます。その後、バックチャンバーを傾けて、ベントキャップの蓋を開きます。インキュベーターでCF - 4を置きます。 </li><li> CF - 4はコンフルエントになるまで、少なくとも週1回の培地の20％を変更します。 </li><li>収穫の細胞は、トリプシン/ EDTAを70mlと約200 mlのPBSを使用して）Bに説明。 </li></ol>駐在結果この分離法を用いて、我々は、12日目で一次コロニー形成を観察した。以前の研究では4.0の平均値±末梢血中の各MLから派生した0,8 ECFC -コロニーを回復することができた4 ECFCsは、典型的な石畳の外観を示した。大規模な展開の後、100細胞/ cm 2の播種密度で、我々は3雄と雌1匹のボランティア（26と50歳の間の年齢）から約30日間の合計で&gt; 1 × 10 8細胞を得た。 このような条件下で培養ECFCは、増殖、機能およびゲノム的に安定であることが示された4さらに細胞をさらに使用するために液体窒素で（10％DMSOで）保存することができます。3,4

<ol>
	<li>Solovey, A., Lin, Y., Browne, P., Choong, S., Wayner, E., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+activated+endothelial+cells+in+sickle+cell+anemia.">Circulating activated endothelial cells in sickle cell anemia.</a> N Engl J Med. 337, 1584-1590 (1997).</li><li>Yoder, M. C., Mead, L. E., Prater, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+large-scale+expansion+of+functional+mesenchymal+stem+cells+from+unmanipulated+bone+marrow+without+animal+serum.">Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.</a> Tissue Eng Part C Methods. 14, 185-196 (2008).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. , (2009).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J Vis Exp. , (2008).</li></ol>

この小説の分離と拡張メソッドは、任意のセルの分離を（不要密度勾配）回避することで、より効率的で時間がかかり、簡単で効率的なプロセスであり、従来の手法よりも安価。ウシ抗原と推定されるその後のxenoimmunizationにpHPL接触を使用することによって除外されます。培養期間中に小さな血栓がpHPLによって引き起こされる、形​​成することができる。我々の経験に基づいて、これらの?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Endothelial basal Medium-2</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>500ml</td>
</tr>
<tr>
<td>EGM-2 SingleQuots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>EGF, hFGF-B, VEGF, IGF-1, Hydrocortisone, ascorbic acid</td>
</tr>
<tr>
<td>preservative-free Heparin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>&nbsp;</td>
<td>10U/ml per EGM-2</td>
</tr>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>2mM per EGM-2</td>
</tr>
<tr>
<td>Penicillin and Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>100U/mL Penicillin and 100&mu;g/mL Streptomycin per EGM-2</td>
</tr>
<tr>
<td>Trypsin/1mM EDTA </td>
<td>&nbsp;</td>
<td>Gibco Cell Culture, Invitrogen </td>
<td>&nbsp;</td>
<td>7ml for T75 flask 70ml for CF-4</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Four-layered cell factory (CF-4) </td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Funnel</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T75cm2 culture flask (with vented cap)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 mL vacutainer tubes (preservative-free sodium heparin) </td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>preloaded with 108 IU/6ml of preservative-free sodium heparin</td>
</tr>
<tr>
<td>50ml Falcon tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Squeezer</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0,2 pore size sterile filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>2x 500ml per EGM-2</td>
</tr>		</tbody>
		</table>
		</div>

isolation and large scale expansion of adult human endothelial colony forming progenitor cells

本論文では、ヘパリンから内皮コロニー形成前駆細胞（ECFCs）のための新規な復旧戦略を導入し、それ以外は12日間の平均値内に成人ヒト末梢血をunmanipulated。大規模な展開の後に&gt; 1 × 10 8 ECFCsは更なる試験のために取得することができます。有利にpHPLを使用してウシ血清抗原とヒト細胞の接触を排除することができます。フローサイトメトリーおよび免疫組織化学法により単離された細胞はECFCと構造がマトリゲルアッセイで試験することができるように血管を形成するためにin vitroでの機能として特徴づけることができる。さらにこれらの細胞は、皮下、in vivoで機能的、灌流血管を形成するために、マウスモデルに注入することができる。後の長期的な拡大と凍結保存、増殖、機能、ゲノムの安定性が保持されているように見える。3,4 この動物タンパク質フリーの分離と拡張メソッドはECFCsを大量に生成するために容易に適用可能です。

Watch this Scientific Journal Video about Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells at JoVE.com

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

内皮コロニー形成前駆細胞（ECFCs）は血管の恒常性と修復を研究するために有望なツールです。<sup&gt; 1,2</sup&gt;このホワイトペーパーでは、抗ウシ予防接種のリスクを減少させるプールされたヒト血小板溶解液（pHPL）とヘパリン成人ヒト末梢血から分離し、ECFCの拡大のための新しい動物血清フリーの方法を紹介します。

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-large-scale-expansion-adult-human-endothelial-colony

Research

JoVE Journal

Biology

10.6K ビュー.  Medical University of Graz, Austria. 内皮コロニー形成前駆細胞（ECFCs）は血管の恒常性と修復を研究するために有望なツールです。 1,2このホワイトペーパーでは、抗ウシ予防接種のリスクを減少させるプールされたヒト血小板溶解液（pHPL）とヘパリン成人ヒト末梢血から分離し、ECFCの拡大のための新しい動物血清フリーの方法を紹介します。

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

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

ヒト臍帯静脈内皮細胞の単離（HUVEC）

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

生物学

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

メタゲノムライブラリの大規模スクリーン

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

弁内皮細胞の単離

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

鳥類胚弁膜前駆細胞の単離と培養

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube 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 ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

インスリン発現コロニーを形成する前駆細胞の定量アッセイ

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

クローン性増殖のためのCD133 +肝幹細胞の単離

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

ヒト臍帯血由来血管内皮コロニー形成細胞の表現型および機能解析

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

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&amp;#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 ...