この材料はグラント号下の国立科学財団によってサポートされる作業に基づいてください。CMMI 1452943、アーカンソー大学の名誉の大学によって。我々 はまたアーカンソーの臍帯血バンク臍帯血の単位を提供する私たちを認識したいと思います。

アーカンソー制度検討委員会 (承認番号 16-04-722) の大学の承認を得て本研究を行った。クエン酸リン酸ブドウ糖 (CPD) ソリューション アーカンソー臍帯血バンクでの臍帯血の単位が採集し、ストレージの要件を満たしていないユニット研究寄付しました。臍帯血の単位が周囲温度でコレクションの 24 h 内のラボに取った。
 1 臍帯血から血管内皮前駆細胞の分離 <ol><li>試薬の準備。。
	<ol><li>インスリン様成長因子 (IGF) の 20 ng/mL、1 μ G/ml アスコルビン酸、5 ng/mL 組換えひと上皮増殖因子 (rhEGF)、22.5 μ G/ml を添加した内皮細胞基本培地 (EBM) を 10% 牛胎児血清 (FBS) に追加することで EGM を準備ヘパリン、および 0.5 ng/mL 血管内皮増殖因子 (VEGF)、10 ng/mL 組換えひと線維芽細胞成長因子 B の (rhFGF-B)、0.2 μ g/mL ヒドロコルチゾン 2% ペニシリン-ストレプトマイシン-グルタミンと。0.2 μ m ポリエーテルサルホン (PES) 膜の真空フィルターを用いた培養培地を滅菌します</li>。
		<li>準備ラットの尾私コラーゲン液 0.02 N 酢酸を使用して 50 μ g/mL の濃度で。0.2 μ m のシリンジ フィルターを使用してこのソリューションを滅菌します</li>。
		準備されたラットの 2 mL で <li>前コート 6 ウェル プレートは私に各ウェルのコラーゲン溶液を尾します。播種前に 5% CO 2 1 h 以上の 37 ° C で維持されるインキュベーター内に配置します</li>。
		<li>臍帯血とハンクス平衡塩ソリューション (HBSS) 1:1 の濃度の約 25 mL の希釈します</li>。
		<li>準備ラジオ免疫沈澱試金塩化ナトリウム 150 mM、1% トリトン X-100, デオキシ コール酸ナトリウム 0.5%、1 mM の β-グリセロリン酸、0.1 %sds と 50 mM トリス (pH 8.0) を使用して (RIPA) バッファーします</li>。
	</ol></li> <li>血管内皮前駆細胞の単離。
	<ol><li>暖かいすべて準備隔離前の 37 ° C で維持水浴の試薬</li>。
		<li>密度勾配中の試薬の 20 mL を 50 mL の遠心管に追加します</li>。
		<li>慎重にチューブの側の壁に向かってピペットを目指すことでこの層を壊すことがなく密度勾配媒体を含む遠心管中の希薄臍帯血 20 mL をレイヤーします</li>。
		<li>は、オフ遠心ローターのブレーキと、室温で 30 分間 800 × g で遠心分離によって血液の密度 ( 図 1) に基づくコンポーネントを区切ります。異なるレイヤーを邪魔しないでください</li>。
		<li>は、慎重にピペット チップが MNC に到達できるまでのレイヤー層 ( 図 1 を参照)、他のセルを乱すことがなくチューブからそれをピペッティングによるプラズマ層を削除します。削除されたプラズマを破棄します 。
		注: プラズマを削除するピペット チップを使用している間プラズマ MNC 層擾乱を抑える ( 図 1) の上の小さい層のままにします</li>。
		<li>18 g 針に装着する注射器を使用して多国籍企業を含むバフィー コートを収集し、新鮮な遠心管にそれを転送します</li>。
		<li>EBM の等量に追加収集した遠心多国籍企業これらの管 500 × g 10 分間で 4 ° c. 破棄上澄み。 。
		注: 細胞ペレットが表示されます赤い赤血球・赤血球の存在のため</li>。
		<li>は、赤色の血液細胞を溶解する塩化アンモニウム溶液 5 mL を追加します。時折揺れとの 5-10 分のための氷にこのチューブをインキュベートします 。
		注: 注意が必要このステップの間に時間の長さを超えないように多国籍企業に有害することができます</li>。
		<li>(500 g) x 4 ° C で 10 分間のチューブを遠心分離し、上澄みを廃棄します。赤血球が持続する場合手順を繰り返して 1.2.8 と 1.2.9 ペレットでは赤い色は認められなかった</li>。
		<li>準備 EGM でペレットを再懸濁し、多国籍企業をカウントするため、検定を使用します</li>。
		<li>吸引ラット尾私 (ステップ 1.1.3) からのコラーゲン膜 6 ウェル プレート、リン酸緩衝生理食塩水 (PBS) × 1 で 3 回洗浄します</li>。
		<li>種子 1 x 10 7 各ウェルの多国籍企業の濃度のコラーゲン プレートに EGM で再停止される多国籍企業ペレットです。37 ° C、5% CO 2 インキュベーターにそれを配置します</li>。
		<li>後 24 h、培地を吸引、EGM で一度洗浄します。各ウェルに EGM 培地 3 mL を追加し、37 ° C、5% CO 2 インキュベーターに戻した</li>。
		<li>は、毎日 7 日間新鮮な EGM 媒体とプレートを補充します。7 日後 EGM 媒体に他の毎日を変更します</li>。
		<li>血管内皮細胞コロニーの進行を追跡するため、毎日 6 ウェル プレートの画像を取る明視野顕微鏡を用いたします。マーク プレート、コロニーが彼らの成長を追跡するため発生します 。
		注: それは通常文化に表示する植民地、5 ~ 9 日かかります</li>。
	</ol></li> <li>血管内皮前駆細胞の拡張。
	<ol><li>コート T-25 培養用フラスコのラット尾私のコラーゲン (50 μ g/mL) 37 ° C、5% CO 2 インキュベーター、少なくとも 60 分吸引それを置くし、T-25 細胞培養用フラスコ、細胞を播種する前に 1x PBS で 3 回洗う</li>。
		<li>は、各ウェルに 0.05% トリプシン EDTA 溶液を 150 μ l 添加を使用サイズ約 3 mm に達すると内皮細胞コロニーを trypsinize します。6 ウェル プレートを 37 ° C、2-3 分のため 5% CO 2 インキュベーターに配置</li>
		<li>は、添付の細胞を除去する 6 ウェル プレートを軽きます。すぐに EGM の 2 mL を加えて 15 mL 遠心管の細胞を回復します</li>。
		<li>診断を使用してセルをカウントし、初期播種密度を計算します</li>。
		400 x g で 5 分間 <li>スピン 15 mL 遠心管は EGM で細胞ペレットを再懸濁しますで 〜 500,000 細胞 T-25 フラスコをシードします。通路 1 としてこのフラスコをマークし、37 ° C、5% CO 2 インキュベーターに戻って T-25 フラスコを配置します 。
		注: セルは EGM の成長を比較する EBM と DMEM でめっきすることをがします</li>。
		<li>さらに通路のフラスコでは、90% の合流点に達すると、手順 1.3.1 - 1.3.5 と T-75 または T-175 細胞培養フラスコに 10 の 4 セル/cm 2 で細胞を再シードします。隔離されたセル (手順 2 および 3) の評価に進みます</li>。
	</ol></li></ol> 2。隔離されたセルを使用して西部にしみが付く特性 隔離されたセルを特徴付ける拡張手順に従うときすべての通路に <ol><li>追加 50-100 μ L の換散バッファー。約 500,000 以上の細胞から蛋白質を収集します</li>。
	<li>の細胞を破壊し、蛋白質を解放する精力的にピペットを上下。収集し、遠心管にバッファーを転送します</li>。
	<li>は 500 × g で遠心管を回転し、新しい微量遠心チューブに上清を慎重に転送します。ラベル付けし、長期保存用-80 ° c チューブを保存します</li>。
	<li>いずれかのブラッドフォードを使用して各チューブの蛋白質の量を定量化する &#39; s または BCA アッセイ 27 , 28 , 29</li>。
	<li>は、ウエスタンブロット標準手順 27 , 28 , 29 を使用して実行します。総蛋白質 CD31、CD34、VEGFR2、α SMA の表現のための 5 μ g を分析します。データの正規化のため α-チューブリンを使用します</li>。
</ol> 3。間接蛍光抗体法 <ol><li>50% エタノールで 18 mm ガラス coverslips に超音波を照射して乾燥させる。それらを殺菌する光 uv クリーン coverslips を残すバイオ セーフティ フードで一晩 12 ウェル プレートで</li>。
	<li>コート 50 ・ #18 と coverslips1; グラム/mL のラットの尾私のコラーゲンと 37 ° C, 60 分、少なくとも 5% CO 2 インキュベーターにプレートを転送</li>
	<li>は、コラーゲンを吸引し、洗浄、coverslips に 12 ウェル プレートに 1 mL の 1x PBS を追加します。1 × PBS を吸引し、2 回繰り返します</li>。
	<li>シード 250,000 培養も 37 ° C では、免疫染色を行う前に、少なくとも 3 日間 5% CO 2 インキュベーターに戻ってそれらの場所で Epc</li>。
	<li>1x PBS で 3 回洗浄します。各ウェルに冷たいメタノール 1 mL を追加し、セルを修復するのに 15 分間、室温でインキュベートします</li>。
	<li>は、標準プロトコル 27 , 28 , 29 を使用して染色を行います。抗体を使用して、彼らの適切な希薄で (例えば CD31 (1:20)、CD34 (1:50)、α SMA (1: 100) と VEGFR2 (1:50)).</li>
	<li>落射蛍光顕微鏡を用いた immunostained coverslips の画像を撮影します</li>。
</ol>

<ol>
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S. <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+Culture+of+Human+CD133++Non-adherent+Endothelial+Forming+Cells.">Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells.</a> Bio-Protocol. 6 (7), (2016).</li></ol>

著者が明らかに何もありません。

前述したように、付着性の Epc は、石畳の形態を所有しています。私たちの孤立した多国籍企業から進歩した紡錘形細胞コロニー (図 2A 2 D) 初期段階で石畳の植民地 (図 2E 2 階) に 10 日間の培養期間。Epc ラベル付けされている異なる研究グループによって異なる後期の血管内皮前駆細胞の10、としてすなわ...

いくつかの調査官は、特性と人間 Epc5,10、11,12,13の可能性を勉強しています。Epc は、循環血管内皮組織低酸素、虚血、外傷、あるいは腫瘍形成のサイトに従うし、新しい血管構造4,14の形成に貢献する能力を持っている細胞として記述することができます。観測へ生後発生の形での血管新生の関与は、これらの細胞とその治療への応用4,15,の病態生理の理解につながっている16. 心血管の病理学9,15,16,17,と18,19 相関する個々 の Epc の数が示されています。 ,20。他の研究も Epc を弁線維芽細胞のような表現型に分化して組織工学心臓弁7、21のこれらの細胞を使用できることを提案しました。
調査4間の不一致のための Epc を分離するために必要な特定の細胞表面分子が明確に識別されていません。いくつかグループ1,17,22,23, Epc の推定される可能性があることを示唆によって実行された多国籍企業のさまざまな培養条件への露出で、特定のマトリックスへの接着異なる表現型プロパティを表示します。これらのプロパティには、貪食能力、マトリゲル、管形成と Dil アセチル化低密度リポタンパク質の摂取量の不足が含まれます。高いクローンと増殖能は、どの Epc と階層化された5をすることができます 2 つのプロパティです。Epc はまた4ひと胎児肺線維芽細胞の遊走と、体外に尿細管を形成できます。これらの細胞は、血管内皮細胞表面マーカーを表現して造血マーカー13,24,25のいくつかを共有するために知られています。フェノタイピング Epc の広く受け入れられている積極的に表現されたマーカーは CD31 CD34、血管内皮増殖因子受容体 2 (VEGFR2)、フォン ・ ヴィレブランド因子 (vWF)、CD133、c キットと血管内皮細胞のカドヘリン (VE カドヘリン)4,18. 共同 CD90、CD45、CD14、CD115、または α-平滑筋アクチン (α SMA) を発現する細胞が貪食、細菌およびde novo人間を形成することができない限られた増殖、潜在的な能力のため Epc をするとは見なされません船生体内で4,7。この資料では、プロトコルをソートの任意のセルを必要とせず人間の臍帯血から血管内皮前駆細胞の分離のための修正されたプロトコルについて説明します。この記事の負のインジケーターとして α SMA との肯定的なマーカーとして VEGFR2、CD34、CD31 を使いました。
この記事での分離及び細胞を使用して選別することがなく臍帯血由来の血管内皮前駆細胞を培養法特化成長因子 (EGM) と内皮増殖培を提案する.この EGM には、血管内皮増殖因子 (VEGF) と線維芽細胞成長因子 (FGF)、生存、増殖、および内皮細胞26の移行を強化が含まれています。セルの石畳の形態を維持するために責任があるアスコルビン酸も含まれていますインスリン様成長因子-1 (IGF-1 の)、血管新生と渡り鳥関数を提供します。成長因子の長期的な安定性の向上は、中型26ヘパリン。血管内皮細胞の培養液に追加他の成長因子には EGF26 に細胞の感受性を高める、ヒドロコルチゾンと分化、細胞増殖を刺激するのに役立ちます表皮の成長因子 (EGF) の補充が含まれています.この特定の成長媒体の使用の Epc 内皮細胞基本培地 (EBM) またはダルベッコ変更イーグル培地 (DMEM) と比較して高い数値を生成することを示す.

<table><tbody><tr><td>&lt;strong&gt;A) For isolation and culturing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EGM-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td>This product comes with all the growth factors needed to make the Endothelial Growth Medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Pencillin-Streptomycin-Glutamine (100x)</td><td>Thermofisher Scientific</td><td>10378016</td><td></td></tr><tr><td>Ficoll-Paque</td><td>GE Heatlhcare</td><td>17-1440-02</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermofisher Scientific</td><td>14170-112</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Stem Cell Technologies</td><td>7850</td><td></td></tr><tr><td>1x Phosphate Buffer Saline</td><td>Thermofisher Scientific</td><td>14190250</td><td></td></tr><tr><td>Rat Tail I Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Amresco</td><td>0714-500ML</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300054</td><td></td></tr><tr><td>HEPES buffer</td><td>Thermofisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Thermofisher Scientific</td><td>10566-016</td><td></td></tr><tr><td>&lt;strong&gt;B) Antibodies and cell lysates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD31&amp;nbsp;</td><td>Abcam</td><td>ab28364</td><td>1:250 dilution&amp;nbsp; for Western blotting</td></tr><tr><td>CD34</td><td>Santa Cruz Biotechnology</td><td>sc-7045</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-SMA</td><td>abcam</td><td>ab5694</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-tubulin</td><td>abcam</td><td>ab7291</td><td>1:2,500 dilution for Western blotting</td></tr><tr><td>VEGFR2</td><td>abcam</td><td>sc504</td><td>1:100 dilution for Western blotting</td></tr><tr><td>Human umbilical vein endothelial cell lysate</td><td>Santa Cruz Biotechnology</td><td>sc24709&amp;nbsp;</td><td></td></tr><tr><td>Valve interstitial cell lysate</td><td></td><td></td><td>Primary cell line cultured from own lab and lysed with RIPA buffer</td></tr><tr><td>&lt;strong&gt;C) Western blotting and immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x Tris/Glycine/SDS buffer</td><td>Biorad</td><td>161-0772</td><td>Used as running buffer</td></tr><tr><td>10x Tris/Glycine buffer</td><td>Biorad</td><td>161-0771</td><td>Used as transfer buffer</td></tr><tr><td>Immobilon-FL transfer membrane</td><td>Merck Millipore</td><td>IPFL0010</td><td>This is a PVDF transfer membrane that has 45 &amp;micro;m pore size and is mentioned in the protocol as western blot membrane</td></tr><tr><td>4x Laemmli sample buffer</td><td>Biorad</td><td>161-0747</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Biorad</td><td>161-0710</td><td></td></tr><tr><td>10% Criterion TGX precast gel</td><td>Biorad</td><td>5671033</td><td></td></tr><tr><td>Prolong Gold antifade</td><td>Thermofisher Scientific</td><td>P36930</td><td>Used for mounting immunostained coverslips for long term storage</td></tr><tr><td>Methanol</td><td>VWR Analytical</td><td>BDH1135-4LP</td><td></td></tr></tbody></table>

isolation of endothelial progenitor cells from human umbilical cord blood

末梢血血管内皮前駆細胞 (Epc) の存在とその血管関与 Ashara と同僚1によって報告されました。後で、他の人は骨髄2,3から発信された Epc の類似の種類の存在を記載されています。最近では、ヨーダとイングラム臍帯血由来の Epc を示した可能性があった、高い増殖大人周辺から分離されたものと比較して血の4,5,6。生後の発生に関与しているから離れて Epc また組織設計血管や心臓弁構造7、8を作成するための細胞源として約束を示しています。様々 な隔離のプロトコルが存在、内皮細胞および造血のマーカーの助けを借りて、上記のソースから派生した単核細胞 (多国籍企業) のセルの並べ替えや養殖専門内皮増殖とこれらの多国籍企業を含むいくつか媒体、またはこれらの技術9の組み合わせ。ここでは、隔離のためのプロトコルを提案する、Epc を使用しての文化専門血管内皮成長因子、西部にしみが付くことを使用して隔離されたセルの特性評価に続いて immunosorting を使用せず培と免疫染色。

分離と血管内皮前駆細胞の拡張: 総合的なプロトコルを描いた模式図 (図 1) を提供します。次の密度勾配媒体でひと臍帯血の密度勾配遠心法別の血コンポーネント レイヤーが観察されました。コラーゲン治療プレートに多国籍企業を播種時にコロニーの副産物が最初日 5 と 7 (図 2A) の間観察され...

Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

このプロトコルの目的は、臍帯血由来の血管内皮前駆細胞を分離することです。一部のアプリケーションは虚血性疾患の治療、心血管のリスクを持つ患者を識別するためのバイオ マーカーとしてこれらの細胞を使用して、作成組織設計の血管や心臓の弁を構築あります。

ひと臍帯血由来の血管内皮前駆細胞の分離

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

isolation-endothelial-progenitor-cells-from-human-umbilical-cord

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

11.0K ビュー.  University of Arkansas. このプロトコルの目的は、臍帯血由来の血管内皮前駆細胞を分離することです。一部のアプリケーションは虚血性疾患の治療、心血管のリスクを持つ患者を識別するためのバイオ マーカーとしてこれらの細胞を使用して、作成組織設計の血管や心臓の弁を構築あります。

ビデオ: Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

発生生物学

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

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

マウス胚性造血内皮細胞の単離

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

フローの状況下で好中球移住研究におけるヒト臍帯静脈内皮細胞とそれらの使用の分離

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

免疫・感染学

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

臍帯血から前駆B細胞サブセットの分離

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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 ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

ヒト臍帯血由来CD34+造血幹細胞および前駆細胞のパンミエロイド分化

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

健康なボランティアからの内皮前駆細胞の単離および心臓手術後の血清試料の影響を受け彼らの渡り鳥の可能性

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

医学

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 ...

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

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

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

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

ひと臍帯血由来 cd34 陽性造血幹細胞の体外増幅+細胞を用いたバルプロ酸

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