Name: isolation of perivascular multipotent precursor cell populations from human cardiac tissue
Uploaded: 2016-10-08T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue at JoVE.com

The authors wish to thank Shonna Johnston, Claire Cryer, Fiona Rossi and Will Ramsay at the University of Edinburgh and Alison Logar and Megan Blanchard at the University of Pittsburgh for their expert assistance with flow cytometry. We also wish to thank Anne Saunderson and Lindsay Mock for their help with obtaining human tissues. Human adult and fetal heart tissue samples were procured with full ethics permission of the NHS Scotland Tayside Committee on Medical Research Ethics and the NHS Lothian Research Ethics Committee (REC08/S1101/1) respectively. This work was supported by grants from the Medical Research Council (BP), British Heart Foundation (BP), Commonwealth of Pennsylvania (BP), Children's Hospital of Pittsburgh (BP), National Institute of Health R01AR49684 (JH) and R21HL083057 (BP), and the Henry J. Mankin Endowed Chair at University of Pittsburgh (JH). JEB was supported by a British Heart Foundation Centre of Research Excellence doctoral training award (RE/08/001/23904). WC was supported in part by an American Heart Association predoctoral fellowship (11PRE7490001).

人間の心臓のサンプルの1処理<ol><li>すべての流体、コンテナ、機器、および専用の操作領域が無菌であることを確認してください。 </li><li> 20％ウシ胎児血清（FBS）、氷上で1％ペニシリン - ストレプトマイシン（P / S）を含む冷却したダルベッコ改変イーグル培地（DMEM）から成る記憶媒体（組織バンクまたは外科チームによって調達）心臓組織試料を置き輸送3。 </li><li>記憶媒体か?...

<ol>
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増加する証拠は、損傷後の成人の心臓の限られた再生能力をサポートしています。傷ついた心の中で、このような回生応答を担当するネイティブ前駆細胞の同定および特徴付けは、関連機構とシグナル伝達経路の理解と治療的にこれらの細胞を利用するアプローチの開発の両方のために重要です。 以前のプロトコルでは、ヒト骨格筋25から血管周囲の前駆細胞サ?...

心臓は長い有糸分裂後の臓器と考えられてきました。しかし、最近の研究では、成人の心臓1で制限された心筋細胞のターンオーバーの存在を実証しました。心筋細胞の分化能を持つネイティブ幹/前駆細胞はまた、cardiosphere形成、SCA-1 +、c-キット+を含め、大人の齧歯動物と人間の心の中に心筋内で同定され、そして最近でてきた、血管周囲の前駆細胞2,3。これらの細胞は、細胞移植またはインサイチュ増殖の刺激を介して心臓の修復/再生を増強することを目的とした治療のための魅力的な候補を表します。 間葉系幹/間質細胞（MSC）は、MSCの治療適用の4,5臨床試験は、心血管修復6、移植片対宿主病7のような複数の病理学的条件のために行われているほとんどすべてのヒト組織から単離されています</sup&gt;、および肝硬変8。有益な効果は、へのMSCの能力に起因している：炎症9のサイトへの家。異なる細胞型10に分化。プロ修復分子11を分泌します 。そして、宿主免疫応答12を調節します 。 MSCの単離は、伝統的に、プラスチック基板への優先的な遵守に依存してきました。しかし、得られた細胞の集団は、一般的に13著しく不均一です。から（ - / CD34 - / CD45 - / - CD56 CD146 + / CD31）キーの血管周囲細胞マーカーの組み合わせで選別（FACS）、蛍光活性化細胞を使用することにより、我々は、多能MSCのような前駆体集団を単離および精製することができました大人の骨格筋や白色脂肪14を含む複数のヒト組織。 様々な非心臓組織における血管周囲の細胞集団は、幹細胞/前駆細胞の特性を有することが示されていますNDは、心血管の設定で臨床使用のために検討されています。周皮細胞、最もよく知られている血管周囲細胞サブセットの一つは、新しい血管15の開発を含むいくつかの病態生理学的役割を果たして不均一な集団であり、血圧16、及び血管の完全17,18の維持の調節。複数の組織に示すように、周皮細胞の特定のサブセットは、ネイティブMSC抗原を発現し、FACS精製後に14初代培養でのMSCのような表現型を維持します。また、これらの細胞を安定に培養内での長期的な表現型を維持したMSC 19,20と同様の多系列分化能を示します。これらの結果は、周皮細胞は、とらえどころのないMSC 14の起源の一つであることを示唆しています。周皮細胞の治療可能性は、虚血負傷者への移植後の心筋瘢痕化および強化された心機能の低下と実証されています心21。最近、我々は成功し、ヒト心筋から周皮細胞を精製し、骨格筋形成3の不在で、そのMSCのような表現型および多分化（脂肪生成、軟骨形成および骨形成）を実証しました。また、心筋周皮細胞が他の臓器から精製されたカウンターパートと比較した場合、差動心筋電位と血管新生能力を示しました。 多能血管周囲の幹/前駆細胞、外膜細胞の第二集団は、陽性のCD34発現22に基づいて、ヒト伏在静脈から単離されています。静脈外膜細胞はクローン原性ポテンシャル、中胚葉分化能およびインビトロでの血管新生促進の可能性を有することが示されています。マウスの虚血損傷した心臓へのこれらの細胞の移植は、間質性線維症の減少、血管新生および心筋の血流の増加、減少、心室DILをもたらしエーション、および増加した心臓の駆出率23。興味深いことに、脂肪外膜細胞が刺激24と周皮細胞の表現型の採用を示唆し、CD34発現を失い、アンジオポエチンII処理に応答して、培養中のCD146の発現をアップレギュレートすることが示されています。心臓内、しかし、外膜の細胞集団は、まだ前向きにFACSにより精製および/または十分に特徴付けられていません。次のセクションで説明した細胞単離手順を用いて、我々は現在、心筋外膜細胞を特徴付けると再生アプリケーションのための彼らの可能性を調査しています。 ここで我々は、ヒト胎児または成人の心筋から血管周囲の幹/前駆細胞の2亜集団を分離し、精製するための方法について説明します。この前向き細胞単離方法は、比較研究やfurtheのためのヒト心臓生検から同質遺伝子血管周囲の幹/前駆細胞サブセットを取得するために研究者を可能にしますrの様々な心臓の病的状態におけるそれらの治療の可能性を探ります。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AbC Anti-mouse Bead Kit</td>
			<td>Molecular Probes</td>
			<td>A-10344</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Gibco</td>
			<td>17100-017</td>
			<td rowspan="3">Reconstitute powder as required and filter sterilise</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Gibco</td>
			<td>17101-015</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>anti-human CD34-PE</td>
			<td>BD Pharmingen</td>
			<td>555822</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD45-APC-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557833</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD56-PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557747</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD144-PerCP-Cy5.5</td>
			<td>BD Pharmingen</td>
			<td>561566</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD146-AF647</td>
			<td>AbD Serotec</td>
			<td>MCA2141A647</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>EGM2-BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>For collection of cells and culture until adhered</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX without sodium pyruvate</td>
			<td>ThermoFischer Scientific</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFischer Scientific</td>
			<td>10500-064</td>
			<td>Freeze in aliquots and keep sterile</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1393</td>
			<td>Dilute with sterile water</td>
		</tr>
		<tr>
			<td>IgG1k-PE</td>
			<td>BD Pharmingen</td>
			<td>559320</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-APC-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557873</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557872</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-PerCP-Cy5.5</td>
			<td>BD Pharmingen</td>
			<td>561566</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-647</td>
			<td>AbD Serotec</td>
			<td>MCA1209A647</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Mouse serum</td>
			<td>Sigma Aldrich</td>
			<td>M5905</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Paraffin Film - Parafilm M</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15979-063</td>
			<td>Freeze in aliquots and keep sterile</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline pH 7.4</td>
			<td>ThermoFischer Scientific</td>
			<td>10010-023</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing Buffer Hybri-Max</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Sigma Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.5%(10X)</td>
			<td>Invitrogen</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;FACSARIA FUSION</td>
			<td>BD Pharmingen</td>
			<td></td>
			<td>Fluorescence Activated Cell Sorter</td>
		</tr>
	</tbody>
</table>

isolation of perivascular multipotent precursor cell populations from human cardiac tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

単一細胞は、前方および側方散乱分布に基づいて破片や二重から区別されました。生細胞は、DAPI染料を取るために彼らの失敗によって同定しました。ゲーティング戦略はこのライブ、全心筋細胞解離（ 図1）のアイソタイプ対照用標識に基づいて選択されました。生きた細胞から、CD45 +細胞が最初にCD56 +細胞に続いて、ゲートアウトしま?...

Watch this Scientific Journal Video about Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue at JoVE.com

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

人間の心臓組織は、心筋再生のために適切であり得る多能血管周囲の前駆細胞集団を保有します。ここに記載された技術は、ネイティブの血管に関連する2つの多能ストローマ細胞集団の同時単離および精製を可能にする、すなわち CD146 + CD34 -周皮細胞およびCD34 + CD146 -外膜細胞、ヒト心筋から。

ヒト心臓組織からの血管周囲多能前駆細胞集団の単離

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

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Developmental Biology

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