本書の指導をしてくれたWei Wang氏、Lingjuan Xu氏、Rong Liu氏、資料の一部を提供してくれたYongyao Tan氏、Bihui Jin氏、Chunxiu You氏、Li Guigang氏、原稿を書いてくれたGuanyu Su氏、原稿の添削をしてくれたXiao Zhou氏、Yihong Xiong氏、Huatao Xie氏、そして完全な指導をしてくれたGuigang Li氏に感謝します。本研究は、中国国家自然科学基金会(第82070936号、81470606年、81570819年)、湖北省保健家族計画科学研究プロジェクト(第1号)の支援を受けて行われました。WJ2017M073)、同済医院のトランスレーショナルメディカル研究プロジェクトトップ10(No.2016ZHYX20)、武漢市若手医療パイオニア育成プロジェクト(No.2015whzqnyxggrc10)、グローバル人材採用プログラム(G2022154028L)、2022年湖北省国家衛生健康委員会プロジェクト(WJ2021ZH0005)、2022年湖北省財政部主題建設財団(42000022815T000000102)

50歳から60歳までのドナーからの辺縁部組織は、赤十字眼科銀行、同済病院(中国、武漢)から入手しました。この議定書は同済倫理委員会によって承認され、ヘルシンキ宣言に従って実施されました。
1. アイソレーション
<ol><li>中期角膜保存培地から辺縁部組織を採取し、超クリーンな作業台で無菌条件下で操作します。</li>
	<li>角膜の周りの虹彩と内皮を、滅菌した外科用丸い刃でこすり落とし、取り除きます。</li>
	<li>角膜と強膜の両側に1mmを残した外科用ブレードで、辺縁組織を12の等しい大きさに切断します。</li>
	<li>辺縁部組織を3.5cmの培養プレートに移し、1 mLのコラゲナーゼA(2 mg/mL、DF-12培地中)を加えて、すべての組織を浸して消化します。</li>
	<li>プレートを37°Cの細胞インキュベーターに入れ、辺縁組織を18時間消化します。 注意: フォローアップ手順は通常、2日目に実行されます。</li>
	<li>5%基底膜マトリックス(マトリゲル)でコーティングされた6ウェル培養プレートを調製します。<ol><li>ウェルの底に5%基底膜マトリックス(MESCMに溶解[表1])をコーティングします。</li>
		<li>200 μL と 1 mL チップ、15 mL 遠心チューブ 1 本、6 ウェルプレート 1 枚を滅菌バッグに入れて調製します。</li>
		<li>滅菌したバッグ(チップと遠心チューブを含む)と新しい6ウェルプレートを-20°Cまたは-80°Cの環境で20分間置きます。基底膜マトリックスを冷蔵庫で4°Cで解凍します。</li>
		<li>-20°Cまたは-80°Cの環境から予冷したチップ、遠心チューブ、6ウェルプレートを取り出し、ウルトラクリーンワークベンチで以下の操作を行います。</li>
		<li>200 μL チップを使用して 5% 基底膜マトリックス 50 μL を 1 mL MESCM に移し、穏やかかつ均一に混合します。</li>
		<li>予冷した 1 mL チップを使用して、5% 基底膜マトリックス(MESCM に溶解)を 6 ウェル培養プレートの 1 つのウェルに移します。</li>
		<li>6ウェルプレートを静かに水平に振とうし、6ウェル培養プレートを37°Cの細胞インキュベーターに約1時間入れてから、次のステップを実行します。</li>
	</ol></li>
	<li>18時間消化後、コラゲナーゼA消化された辺縁組織を取り出すと、目に見える小さな塊と未消化の強膜組織のみが残ります。 注:高倍率顕微鏡では、多くの高密度に充填された細胞(LNCを含む)で構成されるクラスターが明らかになります。</li>
	<li>200 μLのピペットチップを使用して、実体顕微鏡下で未消化の強膜組織からクラスターを剥離します。</li>
	<li>クラスターを3.5cmの培養プレートに移し、0.25%TEに浸漬してから、プレートを細胞インキュベーターに15分間入れます。</li>
	<li>1 mL MESCM と 10% ノックアウト血清を添加して細胞消化を停止し、1 mL のピペットチップを使用してクラスターを個々の細胞に再懸濁するために、静かに上下にピペットで移動します。</li>
	<li>細胞懸濁液を 15 mL 遠心チューブに移し、200 x g で 5 分間遠心分離します。</li>
	<li>細胞沈殿を妨げずに上清を慎重に除去し、1 mL MESCM を添加して細胞を再懸濁します。</li>
	<li>1 mLのピペットチップを使用して、下部の細胞沈殿物がMESCMに均一に懸濁するまで2〜3回ピペットを上下させ、20 μLの細胞懸濁液を採取して細胞をカウントします。</li>
	<li>細胞懸濁液を 1 mL のピペットチップで 5% 基底膜マトリックスコーティング 6 ウェルプレートに移し、MESCM の容量を 2.5 mL に増やします。</li>
	<li>6ウェルプレートを水平に静かに振とうし、細胞を均等に分散させます。</li>
	<li>細胞を画像化して記録した後、培養インキュベーターに移します。細胞を毎日観察して写真に撮り、3〜4日ごとに培地を交換します。</li>
</ol>2. LNCの識別
<ol><li>免疫蛍光染色<ol><li>1 mL 0.25% TE を使用して 37°C のセルインキュベーターで 5 分間、プレートの底部から LNC を消化します。</li>
		<li>1 mL MESCM(ノックアウト血清を含む)を添加して消化を終了し、細胞懸濁液を 200 x g で 5 分間遠心分離し、上清を注意深く吸引します。</li>
		<li>1 mL の MESCM を加えて、細胞を懸濁液に再懸濁します。80 μLの細胞懸濁液(スライドあたり4×103 細胞)を4枚のスライド(20 μL/スライド)用に調製し、遠心分離システムを使用して、メーカーの指示に従って細胞を顕微鏡スライドに沈着させます。</li>
		<li>4%パラホルムアルデヒドで細胞(ステップ3でスライドに接着)を約10分間固定した後、0.2% Triton X-100 in PBSで15分間スライド上の細胞を透過処理します。</li>
		<li>2%ウシ血清アルブミン(BSA)で細胞を1時間ブロックしてから、一次抗体(Pan-CK [1:1000]、Vim [1:100]、CD90 [1:100]、CD105 [1:200]、SCF [1:100]、PDGFRβ [1:100])と4°Cで一晩インキュベートします。 インキュベーション後、スライドをPBST(PBS + 0.1% Tween 20)で洗浄(5分/回、3回)して、結合していない一次抗体を除去します。</li>
		<li>対応する二次抗体(ロバ抗マウスIgG二次抗体TRITC [1:1000]、ロバ抗ウサギIgG FITC [1:500]、ロバ抗マウスIgG 488 [1:300]、ロバ抗ウサギIgG 594 [1:400])を適切なIgG非特異的IgG抗体と1時間インキュベートします。PBST(5分/洗浄、3回)を使用して、結合していない二次抗体を除去します。</li>
		<li>核を 5 μg/mL DAPI(4',6-ジアミジノ-2-フェニルインドール)で対比染色し、スライドを密封します。</li>
		<li>蛍光顕微鏡(倍率400倍;ばく露時間: 50 ms)。</li>
	</ol></li>
	<li>定量的リアルタイムポリメラーゼ連鎖反応(qPCR)<ol><li>RNA Isolation Kit を使用して、製造元の指示に従って全 RNA を抽出します。</li>
		<li>大容量cDNA転写キットを使用して、1〜2μgのトータルRNAをcDNAに逆転写します。</li>
		<li>cDNA(100 ng)、TaqMan Gene Expression Assay Mix (1 μL)、ユニバーサル PCR マスターミックス (10 μL)、ddH 2 O (20 μL まで) を含む 20 μL 溶液中で、逆転写 (42 °C、2分、50 °C、85 分) および qPCR (95 °C、5 分、95 °C、10 秒、40 サイクル) を実行します。</li>
		<li>比較CT法(ΔΔCT)4,9を用いて、相対的な遺伝子発現を調べます。</li>
	</ol></li>
</ol>3. LNCの特性評価
<ol><li>細胞カウント(血球計算盤)<ol><li>プレートの底部から0.25%TEを用いて、37°Cの細胞インキュベーターで5分間LNCを消化します。</li>
		<li>1 mL MESCM(ノックアウト血清を含む)を添加して消化を終了し、細胞懸濁液を 200 x g で 5 分間遠心分離し、上清を注意深く吸引します。</li>
		<li>1 mL の MESCM を加えて細胞を再懸濁し、0.5 mL の遠心チューブで 10 μL の細胞懸濁液を採取します。等量の0.4%トリパンブルー染色溶液を加えます。</li>
		<li>染色した細胞懸濁液10 μLを血球計算盤の計数領域に置き、計数用のカバースリップで覆います。 注:中央の5つの正方形の領域のセル数は「A」として定義されます。1 mLの細胞数は、5A × 104 × 2として計算されます。</li>
	</ol></li>
	<li>フローサイトメトリーによるLNCおよびBMMSCマーカーの検査15<ol><li>フローサイトメトリー15(2-4 × 106イベント/サンプル、各サンプルから2,000個の細胞が得られ、同時にゲーティングされた合計550,000個の細胞)を使用して、LNCおよびBMMSCにおけるSCF、CD73、CD90、およびCD105の発現を検出します。</li>
		<li>キットの説明書に従って、ブロッキングバッファー(3% BSAおよび0.05% Tween-20 in PBS)中で細胞を採取し、染色します。</li>
		<li>フローサイトメーターを用いた蛍光活性化セルソーティング(FACS)解析を実施します。本研究では、FACS DivaソフトウェアとFlowJoソフトウェアを使用した。サンプルごとに 10,000 個のイベントを記録し、生細胞をゲートして解析します。</li>
	</ol></li>
</ol>

ヒト辺縁ニッチ細胞を分離、培養、および同定するためのプロトコール

<ol>
	<li>Le, Q., Xu, J., Deng, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+of+limbal+stem+cell+deficiency.">The diagnosis of limbal stem cell deficiency.</a> Ocular Surface. 16 (1), 58-69 (2018).</li><li>Polisetty, N., Fatima, A., Madhira, S. L., Sangwan, V. S., Vemuganti, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cells+from+limbal+stroma+of+human+eye.">Mesenchymal cells from limbal stroma of human eye.</a> Molecular Vision. 14, 431-442 (2008).</li><li>Xie, H. T., Chen, S. Y., Li, G. G., Tseng, S. C. <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+expansion+of+human+limbal+stromal+niche+cells.">Isolation and expansion of human limbal stromal niche cells.</a> Investigative Ophthalmology &amp; Visual Science. 53 (1), 279-286 (2012).</li><li>Li, G. G., Zhu, Y. T., Xie, H. T., Chen, S. Y., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+derived+from+human+limbal+niche+cells.">Mesenchymal stem cells derived from human limbal niche cells.</a> Investigative Ophthalmology &amp; Visual Science. 53 (9), 5686-5697 (2012).</li><li>Li, G. G., Chen, S. Y., Xie, H. T., Zhu, Y. T., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+potential+of+human+limbal+stromal+niche+cells.">Angiogenesis potential of human limbal stromal niche cells.</a> Investigative Ophthalmology &amp; Visual Science. 53 (7), 3357-3367 (2012).</li><li>Hu, W., Zhang, Y., Tighe, S., Zhu, Y. T., Li, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+isolation+method+of+human+lacrimal+canaliculus+epithelial+stem+cells+by+maintaining+close+association+with+their+niche+cells.">A new isolation method of human lacrimal canaliculus epithelial stem cells by maintaining close association with their niche cells.</a> International Journal of Medical Sciences. 15 (12), 1260-1267 (2018).</li><li>Kumar, A., Xu, Y., Yang, E., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stemness+and+regenerative+potential+of+corneal+stromal+stem+cells+and+their+secretome+after+long-term+storage:+Implications+for+ocular+regeneration.">Stemness and regenerative potential of corneal stromal stem cells and their secretome after long-term storage: Implications for ocular regeneration.</a> Investigative Ophthalmology &amp; Visual Science. 59 (8), 3728-3738 (2018).</li><li>Xiao, Y. T., Qu, J. Y., Xie, H. T., Zhang, M. C., Zhao, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+methods+for+isolation+of+limbal+niche+cells:+Maintenance+of+limbal+epithelial+stem/progenitor+cells.">A comparison of methods for isolation of limbal niche cells: Maintenance of limbal epithelial stem/progenitor cells.</a> Investigative Ophthalmology &amp; Visual Science. 61 (14), 16 (2020).</li><li>Zhu, 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=Limbal+niche+cells+and+three-dimensional+matrigel-induced+dedifferentiation+of+mature+corneal+epithelial+cells.">Limbal niche cells and three-dimensional matrigel-induced dedifferentiation of mature corneal epithelial cells.</a> Investigative Ophthalmology &amp; Visual Science. 63 (5), 1 (2022).</li><li>Basu, S., 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=Human+limbal+biopsy-derived+stromal+stem+cells+prevent+corneal+scarring.">Human limbal biopsy-derived stromal stem cells prevent corneal scarring.</a> Science Translational Medicine. 6 (266), 266ra172 (2014).</li><li>Acar, U., 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=Effect+of+allogeneic+limbal+mesenchymal+stem+cell+therapy+in+corneal+healing:+role+of+administration+route.">Effect of allogeneic limbal mesenchymal stem cell therapy in corneal healing: role of administration route.</a> Ophthalmic Research. 53 (2), 82-89 (2015).</li><li>Katikireddy, K. R., Dana, R., Jurkunas, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+potential+of+limbal+fibroblasts+and+bone+marrow+mesenchymal+stem+cells+to+corneal+epithelial+cells.">Differentiation potential of limbal fibroblasts and bone marrow mesenchymal stem cells to corneal epithelial cells.</a> Stem Cells. 32 (3), 717-729 (2014).</li><li>Polisetti, N., Sharaf, L., Reinhard, T., Schlunck, G. <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+ex+vivo+expansion+of+limbal+mesenchymal+stromal+cells.">Isolation and ex vivo expansion of limbal mesenchymal stromal cells.</a> Bio-Protocols. 12 (14), e4471 (2022).</li><li>Xie, H. T., Chen, S. Y., Li, G. G., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+epithelial+stem/progenitor+cells+attract+stromal+niche+cells+by+SDF-1/CXCR4+signaling+to+prevent+differentiation.">Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation.</a> Stem Cells. 29 (11), 1874-1885 (2011).</li><li>Li, G., 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=Human+limbal+niche+cells+are+a+powerful+regenerative+source+for+the+prevention+of+limbal+stem+cell+deficiency+in+a+rabbit+model.">Human limbal niche cells are a powerful regenerative source for the prevention of limbal stem cell deficiency in a rabbit model.</a> Scientific Reports. 8, 6566 (2018).</li><li>Kumar, A., Yun, H., Funderburgh, M. L., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+therapy+for+the+cornea.">Regenerative therapy for the cornea.</a> Progress In Retinal and Eye Research. 87, 101011 (2022).</li><li>Pineda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Corneal Blindness. Foundations of Corneal Disease. , 299-305 (2020).</li><li>Zieske, J. D., Guimar&#227;es, S. R., Hutcheon, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+keratocyte+proliferation+in+response+to+epithelial+debridement.">Kinetics of keratocyte proliferation in response to epithelial debridement.</a> Experimental Eye Research. 72 (1), 33-39 (2001).</li><li>Resnikoff, S., 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=Global+data+on+visual+impairment+in+the+year+2002.">Global data on visual impairment in the year 2002.</a> Bulletin of the World Health Organization. 82 (11), 844-851 (2004).</li><li>Tan, Y., 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=Limbal+bio-engineered+tissue+employing+3D+nanofiber-aerogel+scaffold+to+facilitate+LSCs+growth+and+migration.">Limbal bio-engineered tissue employing 3D nanofiber-aerogel scaffold to facilitate LSCs growth and migration.</a> Macromolecular Bioscience. 22 (5), e2100441 (2022).</li><li>Aghamirsalim, 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=3D+printed+hydrogels+for+ocular+wound+healing.">3D printed hydrogels for ocular wound healing.</a> Biomedicines. 10 (7), 1562 (2022).</li><li>Sasamoto, Y., Ksander, B. R., Frank, M. H., Frank, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repairing+the+corneal+epithelium+using+limbal+stem+cells+or+alternative+cell-based+therapies.">Repairing the corneal epithelium using limbal stem cells or alternative cell-based therapies.</a> Expert Opinion on Biological Therapy. 18 (5), 505-513 (2018).</li><li>Rohaina, 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=Reconstruction+of+limbal+stem+cell+deficient+corneal+surface+with+induced+human+bone+marrow+mesenchymal+stem+cells+on+amniotic+membrane.">Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane.</a> Translational Research. 163 (3), 200-210 (2014).</li><li>O'Callaghan, A. R., Dziasko, M. A., Sheth-Shah, R., Lewis, M. P., Daniels, J. T. J. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+mucosa+tissue+equivalents+for+the+treatment+of+limbal+stem+cell+deficiency.">Oral mucosa tissue equivalents for the treatment of limbal stem cell deficiency.</a> Advanced Biosystems. 4 (7), e1900265 (2020).</li><li>Yu, D., Chen, M., Sun, X., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+induced+pluripotent+stem+cells+into+corneal+epithelial-like+cells.">Differentiation of mouse induced pluripotent stem cells into corneal epithelial-like cells.</a> Cell Biology International. 37 (1), 87-94 (2013).</li><li>Zeppieri, 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+stem+cells+for+corneal+wound+healing+after+laser-induced+corneal+lesions+in+mice.">Adipose-derived stem cells for corneal wound healing after laser-induced corneal lesions in mice.</a> Journal of Clinical Medicine. 6 (12), 115 (2017).</li><li>Kumar, A., Kumar, V., Rattan, V., Jha, V., Bhattacharyya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretome+cues+modulate+the+neurogenic+potential+of+bone+marrow+and+dental+stem+cells.">Secretome cues modulate the neurogenic potential of bone marrow and dental stem cells.</a> Molecular Neurobiology. 54 (6), 4672-4682 (2017).</li><li>Hayashi, 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=Coordinated+generation+of+multiple+ocular-like+cell+lineages+and+fabrication+of+functional+corneal+epithelial+cell+sheets+from+human+iPS+cells.">Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells.</a> Nature Protocols. 12 (4), 683-696 (2017).</li><li>Guo, 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=Limbal+niche+cells+are+a+potent+resource+of+adult+mesenchymal+progenitors.">Limbal niche cells are a potent resource of adult mesenchymal progenitors.</a> Journal of Cellular and Molecular Medicine. 22 (7), 3315-3322 (2018).</li><li>Wang, W., 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=Differential+gene+expression+between+limbal+niche+progenitors+and+bone+marrow+derived+mesenchymal+stem+cells.">Differential gene expression between limbal niche progenitors and bone marrow derived mesenchymal stem cells.</a> International Journal of Medical Sciences. 17 (4), 549-557 (2020).</li><li>Gonz&#225;lez, S., Deng, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+native+limbal+stromal+cells+increases+the+expansion+efficiency+of+limbal+stem/progenitor+cells+in+culture.">Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture.</a> Experimental Eye Research. 116, 169-176 (2013).</li><li>Funderburgh, M. L., Du, Y., Mann, M. M., SundarRaj, N., Funderburgh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX6+expression+identifies+progenitor+cells+for+corneal+keratocytes.">PAX6 expression identifies progenitor cells for corneal keratocytes.</a> FASEB Journal. 19 (10), 1371-1373 (2005).</li><li>Funderburgh, J. L., Funderburgh, M. L., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+limbal+stroma.">Stem cells in the limbal stroma.</a> Ocular Surface. 14 (2), 113-120 (2016).</li><li>Chen, S. Y., Hayashida, Y., Chen, M. Y., Xie, H. T., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+isolation+method+of+human+limbal+progenitor+cells+by+maintaining+close+association+with+their+niche+cells.">A new isolation method of human limbal progenitor cells by maintaining close association with their niche cells.</a> Tissue Engineering. Part C, Methods. 17 (5), 537-548 (2011).</li><li>Sato, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SnapShot:+Growing+organoids+from+stem+cells.">SnapShot: Growing organoids from stem cells.</a> Cell. 161 (7), 1700-1701 (2015).</li></ol>

どの著者も関連する財務情報開示を行っていません。

角膜の透明性は、通常、角膜間質における小さな線維(直径25〜30 nm)の規則的な配置と分布によって維持され、これは正常な視力に不可欠です16。世界には2億5,300万人の視覚障害者がおり、そのうち3,600万人が17歳の視覚障害者です。世界保健機関(WHO)は、角膜失明を人間の視力に対する最も深刻な危険の1つと見なしており、世界の全失明の5.1%を占めています...

角膜上皮幹細胞欠損症(LSCD)1、角膜上皮再生症(CES)の発生率は、角膜感染症や外傷によりますます緊急性を増しています。適切に治療しないと、CESDは角膜移植を必要とする失明につながる可能性があります。その結果、CESの再生はより重要になってきています。辺縁ニッチ細胞(LNC)と呼ばれる支持細胞のグループがあり、CES機能に不可欠なサポートを提供します。辺縁間質幹細胞は、Polisettyらによって最初に単離され2、Xieら3によって、辺縁の下皮および辺縁の間質に局在するLNCとして同定されました。LNCは角膜縁を支える幹細胞であり、骨髄由来MSC(BMMSC)の機能を持ち、角膜上皮細胞や角膜間質細胞などに成長を誘導する可能性があります3,4,5,6,7。これまでの研究で、LNCの幹細胞の質は、すでに臨床で広く使用されているBMMSC8よりも原始的であることが示されました。LNCは、特にCESDの治療において、MSCに次ぐ実行可能な選択肢になる可能性さえあります。CESの重要な支持細胞であるLNCは、辺縁部の「ニッチ」構造に由来する幹細胞でもあります。LNCは、成熟角膜上皮細胞(MCEC)のCES9への脱分化に重要な役割を果たす可能性があります。しかし、LNCに関する研究はまだ比較的不十分であり、LNCの用語、単離、精製、同定、および特性に関するコンセンサスはありません。一部の研究者は、LNCを辺縁生検由来間質幹細胞10、辺縁間葉系幹細胞11、辺縁線維芽細胞12、および辺縁間葉系間質細胞13と名付けました。LNCの増殖特性は詳細に記述されておらず、科学的および臨床的応用が有望であり、将来的に最も重要な臨床ツールの1つとなる可能性があるため、LNCの単離、精製、同定、および特性を要約する必要があります。
以前の研究14によると、LNCは主に辺縁の下皮と辺縁の間質に存在します。このプロトコルには、コラゲナーゼAを使用して辺縁組織を処理し、LEPCとLNCからなるクラスターを取得し、0.25%トリプシンEDTA(TE)で単一細胞に消化することが含まれます。次いで、LNCを改変胚性幹細胞培地(MESCM)で選択的に培養し、精製した。この論文で報告されているプロトコルは単純で、ヒトLNCを大量に入手するのに高い効率を持っています。
LNCの単離、培養、同定の詳細な手順は、LNC研究に関心のある科学者のためにビデオに記録されており、必要に応じて便利に繰り返すことができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>5&#956;g/mL</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>V900919</td>
			<td>1.25 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Anti-CD73</td>
			<td>Abcam</td>
			<td>ab202122</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>MERCK</td>
			<td>A1933</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CD105</td>
			<td>Proteintech</td>
			<td>67075-1-Ig</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>CD105</td>
			<td>Abcam</td>
			<td>ab114052</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>CD90</td>
			<td>Proteintech</td>
			<td>66766-1-Ig</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>CD90</td>
			<td>Abcam</td>
			<td>ab307736</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Shanghai Lishen</td>
			<td>K1119K4644</td>
			<td>HF90(HT)</td>
		</tr>
		<tr>
			<td>Centrifuge system</td>
			<td>StatSpin&#160;</td>
			<td>StatSpin CytoFuge 12</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10103578001</td>
			<td>2 mg/mL</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss&#160;</td>
			<td>LSM700</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Culture plate</td>
			<td>virya</td>
			<td>3500356</td>
			<td>35 mm</td>
		</tr>
		<tr>
			<td>DME/F-12 1:1 (1x)&#160;</td>
			<td>cytiva</td>
			<td>SH30023.01</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody</td>
			<td>ThermoFisher</td>
			<td>A16016</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) Secondary Antibody</td>
			<td>ThermoFisher</td>
			<td>31568</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>FACS Diva sofware</td>
			<td>BD Biosciences</td>
			<td>Tree Star</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>Becton Dickinson LSRII</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>olympus</td>
			<td>cx31&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma</td>
			<td>G1914</td>
			<td>50 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>MERCK</td>
			<td>Z359629</td>
			<td>Bright-Line</td>
		</tr>
		<tr>
			<td>High-capacity cDNA Transcription Kit</td>
			<td>ThermoFisher</td>
			<td>4374966</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase-contrast microscope</td>
			<td>&#160;UOP</td>
			<td>DSZ2000X</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS (insulin, transferrin, sodium selenite)</td>
			<td>Sigma</td>
			<td>I3146</td>
			<td>5 &#956;g/mL insulin, 5 &#956;g/mL transferrin, 5 ng/mL sodium selenite</td>
		</tr>
		<tr>
			<td>KnockOut SR Serum Replacement for ESCs/iPSCs</td>
			<td>gibco</td>
			<td>10828-028</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BioCoat</td>
			<td>356234</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pan-CK</td>
			<td>Abcam</td>
			<td>ab7753</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>NoninBio</td>
			<td>NBS0135</td>
			<td>4.00%</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MKBio</td>
			<td>MM-1505</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>PDGFR&#946;</td>
			<td>Abclonal</td>
			<td>A1444</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Real-time fluorescence quantitative PCR instrument</td>
			<td>Applied Biosystems</td>
			<td>Step One Plus</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>4 ng/mL</td>
		</tr>
		<tr>
			<td>Recominant Human Leukemia Inhibitory Factor(Lif)</td>
			<td>Peprotech</td>
			<td>300-05</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>RNeasy Mini RNA Isolation Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>-</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>Bioss</td>
			<td>bs-0545R</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>Abcam</td>
			<td>ab52603</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>ZEISS</td>
			<td>SteREO Discovery. V8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical round blade</td>
			<td>Careforde</td>
			<td>29500</td>
			<td>size 10</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Mix</td>
			<td>Applied Biosystems</td>
			<td>4448489</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>MERCK</td>
			<td>X100</td>
			<td>0.20%</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>ThermoFisher</td>
			<td>15250061</td>
			<td>0.40%</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Genview</td>
			<td>GP3108</td>
			<td>0.25%</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>MERCK</td>
			<td>P9416</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ultra Clean Bench</td>
			<td>LaiTe</td>
			<td>LT20200705</td>
			<td>SW-CJ-IFDG</td>
		</tr>
		<tr>
			<td>Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Vim&#160;</td>
			<td>Abcam</td>
			<td>ab92547</td>
			<td>1:100</td>
		</tr>
	</tbody>
</table>

isolation and identification of limbal niche cells

ここでは、辺縁ニッチ細胞(LNC)の単離と同定のための標準的な手順を報告します。アイバンクから得られた辺縁部組織をLNCの単離に使用しました。組織を無菌条件下で12個に分割し、コラゲナーゼAを使用して細胞培養インキュベーターで37°Cで18時間消化し、LNCおよび辺縁上皮前駆細胞を含む細胞クラスターを得ました。細胞クラスターを0.25%トリプシン-EDTAを用いて37°Cで15分間消化し、単一細胞を得た後、5%マトリゲルでコーティングされたプラスチック表面上の改変胚性幹細胞培地(MESCM)で培養しました。細胞は70%のコンフルエンスで継代し、免疫蛍光法、リアルタイム定量PCR(qPCR)、およびフローサイトメトリーを用いてLNCを同定しました。一次LNCを単離し、12回以上継代した。P4からP6にかけてのLNCの増殖活性が最も高かった。LNCは、BMMSC(SCF、Nestin、Rex1、SSEA4、CD73、CD90、MSX1、P75NTR、およびPDGFRβ)よりも高い幹細胞マーカーを発現していました。さらに、P4 LNCはVIM、CD90、CD105、PDGFRβを均一に発現するが、LNCの同定マーカーとして使用できるPan-CKは発現しないことが示された。 フローサイトメトリー解析では、LNCの約95%、97%、92%、および11%がそれぞれCD73、CD90、CD105、およびSCFを発現し、BMMSCでは68%、99%、20%、および3%であることが示されました。LNCの単離と同定のための標準的なプロセスは、LNCを広く使用するための信頼できる実験室基盤を提供する可能性があります。

LNCの成長 LNCは、上記のように、角膜強膜縁組織のコラゲナーゼA(2 mg/mL)消化の消化法に従って正常に単離されました(図1)。以前に報告された研究3と同様に、コラゲナーゼA消化後、イモムシのようなクラスターが顕微鏡下で可視化されました(図2)。紡錘体細胞の割合は、細胞の継代とともに徐々に増加しました。紡?...

Watch this Scientific Journal Video about Standardizing Limbal Niche Cell LNC Isolation and Characterization to Support Widespread LNC Research at JoVE.com

著者スポットライト:広範なLNC研究をサポートするためのLimbal Niche Cell(LNC)の単離と特性評価の標準化(英語) 

ここでは、ヒトの辺縁ニッチ細胞を単離して同定するためのプロトコルを紹介します。

辺縁ニッチ細胞の単離と同定

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

私たちの研究は、辺縁幹細胞欠損症を治療し、患者の視力を回復するために、辺縁ニッチの再構築に焦点を当てています。私たちは、辺縁ニッチ細胞が辺縁幹細胞の機能を支える重要な細胞であるのかどうか、もしそうなら、それがどのように機能するのかを解明しようとしています。最近の研究では、骨髄からの間葉系幹細胞の結膜下移植により、アルカリ焼けした眼の辺縁幹細胞欠損症が予防されることが報告されています。現在、私たちの研究を進めるために使用されている技術の1つは、辺縁ニッチ細胞の特性評価のために単一の細胞レベルで主要なシグナル伝達経路の活性を解析できるシングルセルシーケンシングです。LNCは将来的に重要な臨床ツールになる可能性を秘めていますが、その成長特性はあまり詳細に検討されていません。それらの有望な科学的および臨床的応用を考慮すると、LNCの単離、精製、同定、および特性を要約する必要があります。

isolation-and-identification-of-limbal-niche-cells

Research

JoVE Journal

Medicine

Isolation and Identification of Limbal Niche Cells

679 ビュー.  Huazhong University of Science and Technology. 私たちの研究は、辺縁幹細胞欠損症を治療し、患者の視力を回復するために、辺縁ニッチの再構築に焦点を当てています。私たちは、辺縁ニッチ細胞が辺縁幹細胞の機能を支える重要な細胞であるのかどうか、もしそうなら、それがどのように機能するのかを解明しようとしています。最近の研究では、骨髄からの間葉系幹細胞の結膜下移植により、アルカリ焼けした?...

ビデオ: 著者スポットライト:広範なLNC研究をサポートするためのLimbal Niche Cell(LNC)の単離と特性評価の標準化(英語) 

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

カルボキシフルオレセインスクシンイミジルエステル（CFSE）を用いてヒト神経膠芽腫のスロー非分裂細胞の同定および単離

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

非接触型誘電泳動によりラベルフリーの単離と細胞の濃縮

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

腫瘍特異的内皮細胞の単離および培養拡大

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

マウスで輪部幹細胞欠損を作成するための単純な機械的手順

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

犬の動脈と静脈からの初代内皮細胞の単離および培養

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

ラットにおける骨髄間葉系幹細胞の単離と培養

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

血管応答を調べるための肺内動脈および平滑筋細胞の分離

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

マウスの精密アルカリ熱傷のための修正生検パンチ法

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

著者スポットライト:角膜混濁の治療と再生の研究の進歩 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

マウスモデルにおけるパンチトレフィン法を用いた角膜および辺縁部のアルカリ損傷誘導

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...