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...

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><tbody><tr><td>1.7 mL microcentrifuge tube</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>13 mL culture tube</td><td>Thermo Fisher Scientific</td><td>50-809-216</td><td></td></tr><tr><td>15 mL conical tube</td><td>Greiner Bio-one</td><td>188 271</td><td></td></tr><tr><td>5 mL test tube with cell strainer snap cap</td><td>Thermo Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>50 mL conical tube</td><td>Greiner Bio-one</td><td>227 261</td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Thermo Fisher Scientific</td><td>22-363-548</td><td></td></tr><tr><td>Anti-CD31-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-519</td><td></td></tr><tr><td>Anti-CD45-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-491</td><td></td></tr><tr><td>Anti-Sca1-APC antibody</td><td>Miltenyi Biotec</td><td>130-102-833</td><td></td></tr><tr><td>Anti-Ter119-FITC antibody</td><td>Miltenyi Biotec</td><td>130-112-908</td><td></td></tr><tr><td>BSA</td><td>Gold Biotechnology</td><td>A-420-500</td><td></td></tr><tr><td>Collagenase type II</td><td>Thermo Fisher Scientific</td><td>17101-015</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190-144</td><td></td></tr><tr><td>F-12 medium</td><td>Thermo Fisher Scientific</td><td>11765-054</td><td></td></tr><tr><td>FcR blocking reagent</td><td>Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>Hanks&#039; balanced salt solution (HBSS)</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>16050-122</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Propidium iodide solution</td><td>Miltenyi Biotec</td><td>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。
<p class="jo...

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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

Research

JoVE Journal

Biology

5.6K 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 Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

天井の培養における成熟脂肪細胞の脱分化を経てヒト脂肪幹細胞の生成

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

発生生物学

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

ブタ皮下脂肪組織から分離し、脂肪由来幹細胞の分化

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

<meta charset="utf8"></head><body>トランスクリプトームおよびエピゲノム解析のためのマウス白色脂肪細胞の単離とプロセシング

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

トランスクリプトームおよびエピゲノム解析のためのマウス白色脂肪細胞の単離とプロセシング

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

遺伝学

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

分離、拡張、および CD31 cd34 陽性 + + ひと大網と皮下脂肪組織から血管内皮細胞の脂肪分化誘導

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

免疫・感染学

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

マウス腹腔内脂肪貯蔵所からの脂肪形成性および線維炎症性間質細胞亜集団の単離

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

新生児マウスからの白色および褐色の前読細胞の分離と分化

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

生物学

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

初代培養における分化マウス脂肪細胞:インスリン抵抗性のモデル

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

マウス血管周囲脂肪組織から単離された前駆脂肪細胞の培養

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

著者スポットライト:マウス血管周囲脂肪組織からのSVF由来前脂肪細胞の単離、培養、および脂肪形成誘導の意義 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

マウス上端体脂肪組織からの脂肪前駆細胞の調製

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天井の培養における成熟脂肪細胞の脱分化を経てヒト脂肪幹細胞の生成

ブタ皮下脂肪組織から分離し、脂肪由来幹細胞の分化

トランスクリプトームおよびエピゲノム解析のためのマウス白色脂肪細胞の単離とプロセシング

トランスクリプトームおよびエピゲノム解析のためのマウス白色脂肪細胞の単離とプロセシング

分離、拡張、および CD31 cd34 陽性 + + ひと大網と皮下脂肪組織から血管内皮細胞の脂肪分化誘導

マウス腹腔内脂肪貯蔵所からの脂肪形成性および線維炎症性間質細胞亜集団の単離

新生児マウスからの白色および褐色の前読細胞の分離と分化

初代培養における分化マウス脂肪細胞:インスリン抵抗性のモデル

マウス大動脈周囲脂肪組織からの間質血管分画由来前駆脂肪細胞の単離・培養・脂肪誘発

マウス大動脈周囲脂肪組織からの間質血管分画由来前駆脂肪細胞の単離・培養・脂肪誘発