我々は、FACS選別の支援のためのメイヨークリニック顕微鏡細胞分析コアフローサイトメトリー施設を認める。

すべての動物実験手順は、メイヨークリニックの施設動物ケアおよび使用委員会の承認を得て行われました。
1. ソリューションの準備 
<ol><li>コラゲナーゼII 2 gを100 mLハンクスバランス塩溶液(HBSS)に溶解して2%(w/v)溶液を調製します。アリコート 200 μL 各用途に適しています。</li>
	<li>84 mL F-12培地、15 mL馬血清、および1 mLペニシリン/ストレプトマイシンを混合して中和培地を調製します。</li>
	<li>500 mgのウシ血清アルブミン(BSA)と400 μLの0.5 M EDTAをダルベックコのリン酸緩衝生理食塩基(DPBS)の100 mLに溶解してフローサイトメトリーバッファーを調製します。</li>
</ol>2. 上端体脂肪パッドの解離と組織解離
<ol><li>人道的に安楽死させる(イオブルラン/子宮頸部脱臼)1つの雄マウス。このプロトコルでは、4か月のFVBマウスが使用されました。</li>
	<li>腹部に70%エタノールを塗布/スプレーし、きれいなはさみと鉗子を使用して下腹腔を露出させます。</li>
	<li>精巣上体脂肪パッド(近位/精巣に付着)を見つける。精巣と上体脂肪パッドの間の接合部を鈍い鉗子で保持し、優しく引っ張って上体脂肪パッドを解放する。</li>
	<li>5 mL の HBSS ではさみと精巣上体脂肪パッドを使用して切断して精巣を取り除き、50 mL の円錐管で 15 分間の温度で BSA を補います。</li>
	<li>遠心分離機 150 x g で 4 °C で 7 分間</li>
	<li>50 mL円錐管から浮遊精液脂肪パッドを取り、きれいなはさみを使用して組織を細かくミンチ。</li>
	<li>200 μL コラゲナーゼ 2% 溶液を 13 mL の培養チューブで 3.8 mL の HBSS に加えます。ひき肉を加え、37°Cで1時間5rpmで回転インキュベーターにインキュベートします。 回転インキュベーターの代わりに、37°Cでの細胞培養インキュベーターを、時折攪拌して使用することができる。</li>
	<li>内容物全体を50 mL円錐形チューブに移し、10mLの中和媒体を加えます。チューブを2~3回反転して軽く混ぜます。</li>
	<li>70 μmのセルストレーナーを取り付けた別の 50 mL 円錐形チューブを準備します。細胞のストレーナーを使用して、消化された組織を新しいチューブにフィルターします。</li>
	<li>フロースルーを15mLの円錐形チューブに移し、遠心分離機を350 x g で4°Cで10分間移動します。</li>
	<li>上清を慎重に取り除き、5 mLのDPBSで細胞ペレットを再懸濁します。遠心分離機 350 x g で 4 °C で 10 分間</li>
	<li>上清を慎重に取り除き、50 μLのフローサイトメトリーバッファーを追加します。穏やかにピペットでよく混ぜ、氷の上に細胞を保ちます。細胞ペレットの体積と残留上清の量が少ないため、フローサイトメトリーバッファー内の細胞の総体積は50μL(約100 μL)を超えます。</li>
</ol>抗体標識と蛍光活性化細胞分類(FACS)
<ol><li>穏やかなピペットで細胞をよく混合した後、細胞懸濁液の54 μLを1.7 mLマイクロ遠心チューブに移します。6 μLのFcRブロッキング試薬を加え、軽くピペットでよく混ぜます。4°Cで10分間インキュベートします。54 μLを取った後に残った細胞を保存し、ステップ3.5およびステップ3.6で未染色コントロールとして使用するために氷の中に保管します。</li>
	<li>ステップ3.1のインキュベーション中に、抗Sca1-APC、抗テル119-FITC、抗CD31-FITC、抗CD45-FITC抗体をマイクロ遠心チューブに組み合わせて抗体カクテルを調製します。穏やかなピペットでよく混ぜ、光から保護された氷の上に保ちます。</li>
	<li>FcRブロッキング試薬を10分間インキュベートした後、40 μLの抗体カクテルを加え、穏やかなピペットでよく混ぜます。4°Cで10分間インキュベートします。</li>
	<li>染色された細胞に、1 μg/mL の DAPI を加えたフローサイトメトリーバッファーを 500 μL 加えます。セルストレーナースナップキャップを用いた5mL試験管を使用して、穏やかなピペットとフィルターセルでよく混ぜます。細胞を氷の上に置いておきなさい。</li>
	<li>ステップ3.1の残りの細胞に500μLのフローサイトメトリーバッファーを加え、穏やかなピペットでよく混ぜます。5 mL の試験管を使用して、セルストレーナー スナップ キャップを使用してセルをフィルター処理します。これらの細胞を氷の上に保管し、次の手順で未染色のコントロールとして使用します。</li>
	<li>FACS分析または選別器具に染色された細胞と未染色の制御を取ります。<ol>
<li>図 1に示すギャティング戦略を使用して、APC+/FITC-/DAPI-母集団を特定し、分離します。これらの細胞は、生存可能なSca1+ /Ter119-/CD31- /CD45- 細胞を表す。残骸と細胞の凝集体は、前方(FSC)および側面散布(SSC)プロットを使用して枯渇します。次に、DAPI-母集団がゲートされ、続いてAPC+/FITC-集団のゲーティングが続きます。不染色コントロールは、ギャティングパラメータの設定を支援するために使用する必要があります。</li>
		<li>細胞を1.5 mLマイクロ遠心分離チューブ内のフローサイトメトリーバッファーの500 μLに回収します。今後、汚染を最小限に抑えるために、すべての手順を無菌条件下で行う必要があります。1つのマウスから約50,000~10万個の細胞が採取されます。</li>
	</ol>
</li>
	<li>細胞の生存率を評価するために、ヘモサイトメーターを使用して細胞を数える。細胞凝集体はさらなる下流分析を妨げる可能性があるため、細胞凝集体の存在も評価され、これらの凝集体の存在を最小限に抑えます(生存可能な細胞数の5%)。</li>
</ol>

<ol>
	<li>Krotkiewski, M., Bjorntorp, P., Sjostrom, L., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+obesity+on+metabolism+in+men+and+women.+Importance+of+regional+adipose+tissue+distribution.">Impact of obesity on metabolism in men and women. Importance of regional adipose tissue distribution.</a> The Journal of Clinical Investigation. 72, 1150-1162 (1983).</li><li>Salans, L. B., Horton, E. S., Sims, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+obesity+in+man:+cellular+character+of+the+adipose+tissue.">Experimental obesity in man: cellular character of the adipose tissue.</a> The Journal of Clinical Investigation. 50, 1005-1011 (1971).</li><li>Trayhurn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+adipose+tissue+function+and+dysfunction+in+obesity.">Hypoxia and adipose tissue function and dysfunction in obesity.</a> Physiological Reviews. 93, 1-21 (2013).</li><li>Halberg, N., 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=Hypoxia-inducible+factor+1alpha+induces+fibrosis+and+insulin+resistance+in+white+adipose+tissue.">Hypoxia-inducible factor 1alpha induces fibrosis and insulin resistance in white adipose tissue.</a> Molecular and Cellular Biology. 29, 4467-4483 (2009).</li><li>Upadhyay, J., Farr, O., Perakakis, N., Ghaly, W., Mantzoros, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+as+a+Disease.">Obesity as a Disease.</a> Medical Clinics of North America. 102, 13-33 (2018).</li><li>Cawthorn, W. P., Scheller, E. L., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+stem+cells+meet+preadipocyte+commitment:+going+back+to+the+future.">Adipose tissue stem cells meet preadipocyte commitment: going back to the future.</a> Journal of Lipid Research. 53, 227-246 (2012).</li><li>Cho, D. S., Lee, B., Doles, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refining+the+adipose+progenitor+cell+landscape+in+healthy+and+obese+visceral+adipose+tissue+using+single-cell+gene+expression+profiling.">Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.</a> Life Science Alliance. 2, 201900561 (2019).</li><li>Burl, R. B., 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=Deconstructing+Adipogenesis+Induced+by+beta3-Adrenergic+Receptor+Activation+with+Single-Cell+Expression+Profiling.">Deconstructing Adipogenesis Induced by beta3-Adrenergic Receptor Activation with Single-Cell Expression Profiling.</a> Cell Metabolism. 28, 300-309 (2018).</li><li>Hepler, C., 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=Identification+of+functionally+distinct+fibro-inflammatory+and+adipogenic+stromal+subpopulations+in+visceral+adipose+tissue+of+adult+mice.">Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.</a> eLife. 7, 39636 (2018).</li><li>Merrick, D., 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=Identification+of+a+mesenchymal+progenitor+cell+hierarchy+in+adipose+tissue.">Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.</a> Science. 364, 2501 (2019).</li><li>Schwalie, P. C., 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+stromal+cell+population+that+inhibits+adipogenesis+in+mammalian+fat+depots.">A stromal cell population that inhibits adipogenesis in mammalian fat depots.</a> Nature. 559, 103-108 (2018).</li><li>Raajendiran, A., 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=Identification+of+Metabolically+Distinct+Adipocyte+Progenitor+Cells+in+Human+Adipose+Tissues.">Identification of Metabolically Distinct Adipocyte Progenitor Cells in Human Adipose Tissues.</a> Cell Reports. 27, 1528-1540 (2019).</li><li>De Matteis, R., 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=In+vivo+physiological+transdifferentiation+of+adult+adipose+cells.">In vivo physiological transdifferentiation of adult adipose cells.</a> Stem Cells. 27, 2761-2768 (2009).</li><li>Hanamsagar, R., 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=An+optimized+workflow+for+single-cell+transcriptomics+and+repertoire+profiling+of+purified+lymphocytes+from+clinical+samples.">An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples.</a> Scientific Reports. 10, 2219 (2020).</li><li>Marinovic, M. P., 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=Crotamine+induces+browning+of+adipose+tissue+and+increases+energy+expenditure+in+mice.">Crotamine induces browning of adipose tissue and increases energy expenditure in mice.</a> Scientific Reports. 8, 5057 (2018).</li><li>Takahashi, H., 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=Biological+and+clinical+availability+of+adipose-derived+stem+cells+for+pelvic+dead+space+repair.">Biological and clinical availability of adipose-derived stem cells for pelvic dead space repair.</a> Stem Cells Translational Medicine. 1, 803-810 (2012).</li><li>Cowan, C. M., 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=Adipose-derived+adult+stromal+cells+heal+critical-size+mouse+calvarial+defects.">Adipose-derived adult stromal cells heal critical-size mouse calvarial defects.</a> Nature Biotechnology. 22 (5), 560-567 (2004).</li><li>Hagberg, C. E., 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=Flow+Cytometry+of+Mouse+and+Human+Adipocytes+for+the+Analysis+of+Browning+and+Cellular+Heterogeneity.">Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity.</a> Cell Reports. 24, 2746-2756 (2018).</li></ol>

著者らは開示するものは何もない。

単一細胞RNAシーケンシング(scRNA-seq)は、単一細胞レベルで多様な細胞集団を同時に研究する強力なツールとして急速に牽引力を得ています。サンプル調製と高スループットシーケンシングに関連するコストが高いため、実験の成功の可能性を高めるためには、細胞入力(高い生存率と純度)を最適化することが不可欠です。一部の細胞調製プロトコルは、FACSソート14を使用せずに、低スピン化しカラムベースの分離を使用して死んだ細胞および破片の除去に依存している。しかし、これらの方法の多くは、生存可能な回収細胞の数を大幅に減少させ、そして多くの場合、死細胞は完全に除去されない14。このプロトコルは、最小限の細胞残骸、細胞ダブレット、および死細胞を含む非常に生存性の高いSca1+脂肪前駆細胞を分離する簡単な方法を概説する。このプロトコルを使用して分離された細胞は高い生存率を示しましたが、高い生存率を維持するために、細胞の単離とさらに下流分析の間の時間を最小限に抑えることをお勧めします。さらに、高純度のSca1+脂肪前駆細胞を単離するためには、抗体標識が成功することが重要です。したがって、抗体の最適濃度の決定は各抗体に強く推奨され、ステップ3.2における抗体の希釈はそれに応じて調整することができる。
このフローサイトメトリーベースのプロトコルは、特定の脂肪前駆細胞サブ集団における個々の関心に応じてカスタマイズに適している。ここで、代替抗体の組み合わせは、目的の集団を同定し、単離するために使用することができる。実際、広範なAPCマーカーの多様性のために、scRNA-seqを使用してAPCを研究している複数のラボは、他の表面マーカーを使用して細胞を単離するレポートを報告します。例えば、PDGFRA+ /CD44+セル、 CD31-/CD45-/Ter119-/CD29+/CD34+/Sca1+細胞、および Pdgfrb+細胞は下流の APC scRNA-seq 分析8,9,11のために FACS によって単離されている。CD142+亜集団も特徴付けられており、限られた有数の分化能と、脂質生成を抑制する能力を示すことが10,11に示されている。Sca1に加えて、CD55、CD81、およびCD9に対する抗体の組み合わせは、最近、ASCおよび前置体亜集団を前向きに単離し(このプロトコルを使用して)、分子および機能的不均質性7を研究するために使用された。また、インギナス脂肪パッドや褐色脂肪組織を含む他の脂肪パッドから脂肪前駆細胞を脂肪化する前駆細胞は、本プロトコル15、16、17のようなコラゲターゼ解離を用いて単離されているので、本プロトコルは、他の脂肪組織からの脂肪前駆細胞の単離に適用可能であり得る。
現在のアプローチの 1 つの注意点は、未熟な APC の分離に合わせて調整されるということです。本プロトコルは高い生存率APCをもたらすが、他の脂肪組織居住細胞型(すなわち、免疫細胞、線維芽細胞、内皮細胞など)の単離の有効性および効率は不明である。重要なことに、このプロトコルは成熟したアディポサイトの分離には適していません。アディポサイトは、細胞サイズが大きく脆弱性が大きいため、FACSを使用して分離することは非常に困難でした。最近、FACSによる成熟したアディポサイトの単離は、特定のFSC/SSC格子戦略18を用いて大きなノズルサイズ(150μm)および低シース圧(6 psi)を用いて報告された。今後の研究では、この脂肪細胞分離プロトコルとここで説明されているプロトコルを組み合せ、脂肪組織内の多様な細胞型を分離して研究するためのより包括的なパイプラインを確立する可能性があります。

脂肪組織は、エネルギー代謝に重要な役割を果たしています。.過剰なエネルギーは脂質の形で貯蔵され、脂肪組織は栄養状態およびエネルギー需要に応じて有意な膨張または引き込みが可能である。脂肪組織の拡張は、脂肪細胞サイズの増加(肥大)および/または脂肪細胞数の増加(過形成)から生じる可能性があります。後者のプロセスは、脂肪前駆細胞1,2の増殖と分化によって厳しく調節される。肥満の間、脂肪組織は過度に拡大し、低酸素症、炎症、インスリン抵抗性を含む組織機能不全は、しばしば3、4を発症する。これらの合併症は高血圧、糖尿病、心血管疾患、脳卒中、および癌5を含む多くの慢性疾患への危険因子である。したがって、制御されていない脂肪組織の拡張を制限し、脂肪組織の病理を緩和することは、生物医学研究の最優先事項である。脂肪組織の拡張中に、生体脂肪組織由来幹細胞(ASC)は増殖し、順次に前駆細胞(コミット前駆細胞)に増殖し、次いで成熟脂肪細胞に分化する。最近の単細胞RNAシーケンシング(scRNA-seq)研究は、これらの脂肪前駆細胞(APCおよび予後細胞)集団が実質的な分子および機能的ヘテロジニアリティ7、8、9、10、11、12を示していることを示している。例えば、ASCは、減少した有数分化能力を示す一方で、より高い増殖および拡張能力を示す一方で、前置細胞7と比較して。ASCとプリデポサイト集団内でさらなる分子差が報告されるが、これらの違いの機能的関連性は依然として不明である7。これらのデータは、脂肪の前駆細胞プールの複雑さを強調し、これらの重要な細胞集団をよりよく理解し、操作するためのツールを開発し、標準化する必要性を強調しています。
このプロトコルは、単一細胞研究(scRNA-シーケンシング)および細胞培養を含む、敏感な下流分析に適したマウス上端体脂肪パッドからの高生存率Sca1+脂肪前駆細胞集団の分離を詳述する。上端体脂肪パッドの単離および解離は、前述の7,13と同様に、単離されたAPCの生存率を向上させるわずかな改変を伴って行った。簡単に言えば、上端体脂肪パッドからの解離細胞は、Sca1に対する抗体、ASCおよび予読細胞6、7、および他の系統(Lin)マーカーの両方に対する抗体で染色される:Ter119(エリスロイド細胞)、CD31(内皮細胞)、およびCD45(白血球)。生存可能なSca1+/Ter119-/CD31-/CD45-/DAPI-細胞は、蛍光活性化細胞分類(FACS)によってソートされます。重要なことに、このプロトコルは、ASCおよび予後細胞内の機能的に不均一な亜集団を同定した最近の単一細胞RNAシーケンシング研究で報告された、実行可能なSca1+ /Linの脂肪化前駆細胞の分離および分析に成功して検証された。

<table border="0" cellpadding="0" cellspacing="0" style="width:799px;" width="799">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1.7 mL microcentrifuge tube</td>
			<td style="width:181px;">VWR</td>
			<td style="width:119px;">87003-294</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">13 mL culture tube</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-809-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">15 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">188 271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5 mL test tube with cell strainer snap cap</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">08-771-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">50 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">70 &#181;m cell strainer</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD31-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD45-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Sca1-APC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Ter119-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-112-908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">BSA</td>
			<td style="width:181px;">Gold Biotechnology</td>
			<td style="width:119px;">A-420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Collagenase type II</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17101-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">DAPI</td>
			<td style="width:181px;">Thermo Fisher</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">F-12 medium</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">FcR blocking reagent</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hanks&#39; balanced salt solution (HBSS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Horse serum</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Penicillin-streptomycin</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Propidium iodide solution</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-233</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of adipose progenitor cells from mouse epididymal adipose tissues

肥満および代謝障害(糖尿病、心臓病、癌など)は、すべて劇的な脂肪組織リモデリングに関連している。組織に住む脂肪前駆細胞(APC)は脂肪組織恒常性において重要な役割を果たし、組織病理に寄与し得る。単一細胞RNAシーケンシングや単細胞プロテオミクスを含む単細胞解析技術の使用が増え、集団または組織全体の変化の中で個々の細胞発現変化の前例のない解決を可能にすることで、幹細胞/前駆細胞分野を変革しています。本稿では、マウスの上端体脂肪組織を解剖し、単一の脂肪組織由来細胞を分離し、蛍光活性化細胞選別(FACS)を実行して、実行可能なSca1+/CD31 - /CD45-/Ter119- APCを濃縮するための詳細なプロトコルを提供します。これらのプロトコルにより、研究者は単一細胞RNAシーケンシングなどの下流解析に適した高品質のAPCを準備することができます。

この実験では、生後4ヶ月の雄FVBマウスを用いた。FSC/SSCプロットを使用して破片およびダブレットを除外した後、生存可能な細胞(DAPI- 母集団)をゲートし、続いてAPC+/FITC- 母集団の選択を行った(図1)。DAPI、APC、およびFITCのゲートは、未染色の制御に基づいて描かれました。ギャティング戦略を 図 1に示します。
1時間の選別後、分離の質をフローサイトメトリー解析により定量的に評価した(図2、3)。細胞を、生存性染色のためにヨウ化プロピジウム溶液(1:100)で染色した。同じ格子化を使用して、細胞は高い生存率と純度を維持しました:92.6±2.2%単一細胞( 図2の3番目のパネル)、86.4±3.0%生存率(PI- 細胞)、86.0±2.8%APC+/FITC- 細胞(n = 4、標準偏差 ±の平均)これらのパーセンテージは、各ゲート母集団(単一細胞、PI- 細胞、PI-/APC+/FITC- 母集団)の割合として 、それぞれ総細胞数に対する割合として定義された。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61694/61694fig1v2.jpg"> 図1:FACSによる生存可能なSca1+ 脂肪前駆物質単一細胞の単一の分離。 ゲージ戦略は、染色された細胞と未染色のコントロールの両方で示されています。 <a href="https://www.jove.com/files/ftp_upload/61694/61694fig1v2large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61694/61694fig2v2.jpg"> 図2:分離後細胞の生存率を評価するための代表的なフローサイトメトリー分析。 ヨウ化プロピジウムを用いて生存性染色を行った。 <a href="https://www.jove.com/files/ftp_upload/61694/61694fig2v2large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61694/61694fig3.jpg"> 図3:フローサイトメトリー分析における細胞1時間後の分離割合の定量化。単細胞、生存細胞、およびAPC+/FITC-細胞の割合を定量化し、細胞の単離後の純度および生存率を評価した。表示されるデータは、平均±標準偏差を表します。n=4。<a href="https://www.jove.com/files/ftp_upload/61694/61694fig3large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>

Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

蛍光活性化細胞選別を用いて、マウス上体脂肪パッドから生存性の高い脂肪前駆細胞を単離する簡単な方法を提示する。

マウス上端体脂肪組織からの脂肪前駆細胞の調製

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

preparation-adipose-progenitor-cells-from-mouse-epididymal-adipose

Nanyang Technological University

Research

JoVE Journal

Biology

5.5K ビュー.  Mayo Clinic. 蛍光活性化細胞選別を用いて、マウス上体脂肪パッドから生存性の高い脂肪前駆細胞を単離する簡単な方法を提示する。

ビデオ: Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

生物学

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 ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

ヒト大動脈血管周囲脂肪前駆細胞の分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

多様なマウス脂肪デポの同定と解剖

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

血管周囲脂肪組織による変調に焦点を当てた孤立腸間膜動脈を用いた血管緊張応答性の評価

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

メチルサリチル酸除去と3Dイメージングによる脂肪組織構造の探索

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

アカゲザル脂肪由来幹細胞の単離・増殖・分化

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

[18F]FDGマイクロPET/MRイメージングを用いたマウスの褐色およびベージュ色の脂肪組織の可視化と定量化

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

下流アプリケーションのための異なる脂肪デポからの血管内皮細胞の分離と同定(英語)

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

新生仔マウスの肩甲間脂肪組織の間質血管画分からの褐色脂肪細胞の分化とイメージング

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...