我々は感謝し、彼の技術的支援のための彼らの重要な議論とモハマド·レザ·カデムシャリフ博士のレイラShakeri＆博士アレフDournaeiを認める。

1。酵素溶液及び増殖培地（PM）を準備<ol><li> コラゲナーゼI型溶液を作る：コラゲナーゼI型（12 mg / ml）を秤量し、0.2μmのシリンジフィルターを用いて1mlのPBSとフィルターで溶解する。それを15 mlチューブを配置し、必要になるまで-20℃で保管してください。 </li><li> ディスパーゼ溶液を作る：ディスパーゼを秤量（16 mg / ml）と1mlのPBSとフィルターが0.2μmのシリンジフィルターを使用して溶かす。それを15 mlチューブを配置し、必要になるまで4℃で保管してください。 </li><li> 酵素液を作る：1 mlのコラゲナーゼI型溶液（12 mg / ml）と100 mg / mlのペニシリンを含む2 mlの滅菌PBSに1ミリリットルディスパーゼ溶液（16 mg / ml）を、100 mg / mlのストレプトマイシンを追加します。最終体積ディスパーゼとコラゲナーゼIの合計濃度がreceptively 4 mg / mlおよび3 mg / mlであるべきです。次に、4つの15 mlチューブ、それぞれ含む1 mlの酵素液の中でアリコートこのボリューム。各チューブは1パルプ消化を使用することができる。 </li><li> PBSに100 mg / mlのペニシリン、100 mg / mlのストレプトマイシンを追加：洗浄液を作る 。 </li><li> 10％のFBS＆100単位/ mlペニシリンイーグル培地（α-MEM）、100 mg / mlのストレプトマイシンのアルファ改：基本的なメディアを作成します 。 </li><li> 10％のFBS、100μMのL-アスコルビン酸-2 -リン酸、2mM L-グルタミン、100ユニット/ mlペニシリン、100 mg / mlのを補ったイーグル培地（α-MEM）のアルファ改：増殖メディア（PM）を作るストレプトマイシン、0.25 mg / mlのアムホテリシンB </li><li>イーグル培地のアルファ改質（α-MEM）、10％FBS、100μMのL-アスコルビン酸-2 -リン酸、2mM L-グルタミン、100ユニット/ mlペニシリン、100 mg / mlのストレプトマイシン、0.01μMで補足：歯原性メディアを作るデキサメタゾン、5mMのβ-グリセロールリン酸、1.8 mMのリン酸一カリウム。 </li></ol> 2。歯髄幹細胞Isolatio用ヒト歯髄組織を準備N <ol><li>正常ヒト第三大臼歯が承認治験審査委員会（IRB）の下でイマーム·アリの歯科医院で大人（年齢21〜29歳）から収集された。 </li><li>歯は基本培地（α-MEM、10％FBSを添加した）に入れ、4℃で実験室に移した</li><li>無菌状態の下で、バイオハザード、層流フード内で作業して、セットアップが各歯1 100mmペトリ皿を処理することが行われていた。歯の表面を70％エタノールで洗浄した。 </li><li>ペトリ皿の一つで働いている間、歯は抜歯鉗子によって保たれ、手術用メスの刃は、根に破片や歯根膜をきれいにするために使用された。 </li><li>セメント質·エナメル接合はゆっくり髄室を明らかにするために滅菌歯科ディスクを使用して切断した。これは、切削加工、歯科組織の過熱を低減するためにゆっくりと実行しなければならないことを考慮すべきである。 </li><li>歯髄組織を穏やか冠と根から分離した。 </LI&gt; <li>歯髄組織は、手術用メスの刃の助けを借りて、小さな断片に切断した。 </li></ol> 3。ヒト歯髄アイソ<ol><li>歯髄組織は歯髄組織またはhDPSCsの単離のための歯髄組織外植片から直接細胞伸長のいずれかの酵素分解を受けた。 </li><li> 歯髄組織の酵素的解離： <ol><li>歯髄組織の小片を37℃で1時間、酵素溶液中に移した組織を壊すのに役立つ渦30分毎。 </li><li>その後、大きな細胞凝集体を除去し、単細胞懸濁液を70μmのセルストレーナーを介して細胞を通過させることによって得られた。 </li><li>単細胞懸濁液を室温で5分間1200 rpmで遠心分離した。 </li><li>上清を慎重にピペットし、ペレットを1mlの増殖培地（PM）に再懸濁した。 （ 注：増殖培地中のFBSは終了DIS酵素基群集。） </li><li>歯髄の単細胞懸濁液は、PMとの25cm 2の培養フラスコに播種し、次いで、5％CO 2中37℃でインキュベートした。 </li><li>細胞のコンフルエントに達するまで培養培地は3日ごとに変更されました。 </li></ol></li><li> 歯髄組織外植片から直接細胞伸長： <ol><li>歯髄組織が​​1-2mmの断片にみじん切りされ、各部分がPMと25 cmの2培養フラスコに入れた後、37℃、5％のCO 2インチそれは、細胞の伸長のためのPMの総量はさらに、細胞伸長のためのパルプ片（フラスコあたり2〜3 ml）の添付ファイルをサポートしなければならないことを考慮すべきである。 </li><li>伸長が観察された後に培地を変更しました。 </li><li>合流手狭細胞が1:4の割合（通路1）で継代培養した。 </li></ol></li></ol> 4。免疫表現型検査<ol><li> 250,000細胞/通路3内のセルの両方のタイプのチューブを4℃暗所で30分間、PEまたはそれらのアイソタイプとFITC結合抗ヒト抗体でインキュベートした。 （ 注：抗体の濃度は、製造プロトコルに従って適用された） </li><li>インキュベーション時間後、100μlのPBSまたはFACS希釈液を各チューブに添加し、暗所に5分間1,200 rpmで遠心分離した。 </li><li>上清を除去し＆ペレットを100μlのPBSや暗い場所でのFACS希釈液で再懸濁した。 </li><li>標的表面マーカー（CDマーカー）は、BD FACSキャリバーによって評価した。 </li></ol> 5。鉱化細胞へDPSCsの分化誘導とは、アリザリンレッドSアッセイを定量化<ol><li>歯髄組織外植片（DPSC-OG）から歯髄組織の酵素的解離（DPSC-EDとも呼ばれます）または直接的な細胞伸長のいずれかによって得られたDPSCsは3継代継代培養した。 </li><li> Aトン通路3は、細胞を5000細胞/ウェルで6ウェルプレートに種した。 </li><li> 60％の合流点に、歯原性媒体は、従来のPMに取って代わられた。スリーウェルを増殖培地を追加することにより、ネガティブコントロールとして残った。 </li><li>媒体は21日目まで3日ごとに変更します。 </li><li> 21日目に、細胞をPBSで洗浄し、1ml /ウェルを室温で15分間、10％パラホルムアルデヒドで固定した。 </li><li>その後、パラホルムアルデヒドは、慎重にピペットし、細胞を蒸留H 2 Oで3回（各回5〜10分）を洗浄した（ 注：単層を乱さないようにしてください。） </li><li>洗浄した後、1ml /ウェルアリザリンレッドSを室温で20分間添加した。 </li><li>細胞は、任意の追加のアリザリン赤を除去するために脱イオンH 2 O 4倍（優しく揺らしながらたびに、5分間）により洗浄した。 </li><li> 1ml /ウェルH 2 Oは乾燥から細胞を防ぐために追加されました。 </li><li>プレートは視覚insに想定したpectionおよび/または画像取得。 </li><li>アリザリンレッドS定量アッセイのために、H 2 Oを振とうしながら室温で30分間、6ウェルプレート＆インキュベーションの各ウェルに1mlの10％酢酸に置き換えられました。 </li><li>その後、細胞単層/井戸がスクレープ＆酢酸＆細胞の両方であった別の1.5mlマイクロ遠心管に移した。 （ 注：各ウェルは、3つの試験ウェル＆各ED＆OGのグループでは3つの対照ウェルすなわち別のチューブに転送する必要があります） </li><li>チューブは30秒間激しくボルテックスした。 </li><li> 85〜ヒート℃で10分間。 （ 注：蒸発を避けるために、チューブをパラフィルムで密封した。） </li><li>加熱後、試験管を5分間氷上に移した。 （ 注 ：冷却になるまでチューブは開かないでください。） </li><li>スラリーは、15分間20,000 xgで遠心分離した。遠心分離しながら、アリザリンレッド規格が作られたアップ。 </li></ol>アリザリンレッドS標準は、第一希釈緩衝液を希釈＆テーブルに基づいて、2倍希釈系列を作るために使用することにより調製した。 <table border="1"><tbody><tr><td colspan="7"> 染料の高域濃度 </td><td colspan="7"> 染料の低域の濃度 </td></tr><tr><td> 2mMの</td><td> 1mMの</td><td> 500μMの</td><td> 250μM </td><td> 125μMの</td><td> 62.5μM </td><td> 31.3μM </td><td> 15.6μM </td><td> 7.8μM </td><td> 3.9μM </td><td> 1.9μM </td><td> 0.9μM </td><td> 0.4μMの</td><td>ブランク</td></tr></tbody></table><ol start="16"><li>遠心分離後、上清/管1mlの新しい独立した1.5​​mlマイクロ遠心管に移した。 </li><li> pHは〜375μlの10で中和したチューブあたり％水酸化アンモニウム。 （ 注：pHの怒りは4.1から4.5でなければなりません。） </li><li>スタンダード＆サンプル（別々のテスト＆コントロールが含まれています）150μlを96ウェルプレートに不透明な壁、透明な底に添加した。 </li><li>吸光度を405nmで読み取ったと試料からのOD値をさらに分析するための標準的なプロットと比較した。 </li></ol>

<ol>
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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=EF1alpha+and+RPL13a+represent+normalization+genes+suitable+for+RT-qPCR+analysis+of+bone+marrow+derived+mesenchymal+stem+cells.">EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.</a> BMC Mol. Biol. 11, 61 (2010).</li><li>Suchanek, J., 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=Dental+pulp+stem+cells+and+their+characterization.">Dental pulp stem cells and their characterization.</a> Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 153, 31-35 (2009).</li><li>d'Aquino, 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=Human+postnatal+dental+pulp+cells+co-differentiate+into+osteoblasts+and+endotheliocytes:+a+pivotal+synergy+leading+to+adult+bone+tissue+formation.">Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.</a> Cell Death Differ. 14, 1162-1171 (2007).</li><li>Atari, 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=Isolation+of+pluripotent+stem+cells+from+human+third+molar+dental+pulp.">Isolation of pluripotent stem cells from human third molar dental pulp.</a> Histol. Histopathol. 26, 1057-1070 (2011).</li></ol>

特別な利害関係は宣言されません。

このプロトコルは、2つの方法、酵素的解離と歯髄組織外植片からの幹細胞の直接の伸長を用いて歯髄からhDPSCsの単離および特性のプロセスについて説明します。また、象牙芽細胞へのこれらの細胞のインビトロ分化に 、定量的なアリザリンレッドS＆QPCRアッセイにより評価した。 人間の歯から歯髄組織を分離するための既存のプロトコルは、プライヤー（?...

幹細胞は、マルチ効力と自己再生15として知られている2つの顕著な特徴を持っているクローン原性細胞である。異なる複製効力を持つすべての幹細胞のう ​​ち、生後幹細胞などの歯科幹細胞は、それらのアクセシビリティ、可塑性、および他の16の成体幹細胞と比較して高い増殖能力を近年注目を集めている。特徴的には、間葉系幹細胞と同様に、歯髄幹細胞は、複数の分化間充織に能力および/ ​​または in vitro および in vivo の両方において非間葉系統を持って付着クローン原性細胞である。1-8,17,18 DPSCsが特定される彼らの造血抗原の陰性発現（ 例えば 、CD45、CD34、CD14）、およびCD90、CD29、CD73、CD105、CD44およびSTRO-1の陽性発現。19,20 最小痛み＆罹患率との容易な得られた電位は貴重として人間DPSCsを作るMSCのできる源は、骨髄間葉系幹細胞21として普通のソースに比べて。一般的に、DPSCsは伸長法、 すなわち 、歯髄組織外植片からの幹細胞の遊走（DPSC-OG）22から24、および/ ​​または酵素消化によって（DPSC-ED）の4,6,25のいずれかの方法で単離されている。これまでの研究では、単離法と培養条件は 、in vitro の通路26,27 内で異なる集団または系統を誘導できることが示されている。永久歯（pDPSCs）の場合では、Huang らは、酵素消化pDPSCsが手狭DPSCsに比べて高い増殖能力を持っていることを明らかにした。また26は 、乳歯（dDPSCs）の場合、それが実証されたそのSTRO-1とCD34マーカーはdDPSC-OGと比較してdDPSC-EDの続きを表明した。また、dDPSC-EDは、定義された骨/ odonto培地で高い無機化速度が表示されていましたので27によりrのDPSCsの傑出した電位にegenerative薬は、より多くの研究は、異なる分離方法に由来している可能性のある様々な集団をよりよく理解するために必要となります。 ここでは、パルプ抽出のプロセスを容易にするためのワンステップデンタルダイヤモンドディスクを使用することにより、パルプ抽出の簡単な方法を紹介する試みであった。また、EDまたはOGの方法を適用することにより、ヒト歯髄由来幹細胞を単離した後、両群間の一般的なプロパティと分化能についても検討した。

<table border="1"><tbody><tr><td>試薬の名称</td><td>会社</td><td>カタログまたはロット。数</td><td>コメント（オプション） </td></tr><tr><td> α-MEM </td><td> GIBCO </td><td> 11900-073 </td><td></td></tr><tr><td>コラゲナーゼI型</td><td>シグマアルドリッチ</td><td> C0130-100MG </td><td></td></tr><tr><td>ディスパーゼ</td><td> GIBCO </td><td> 17105-041 </td><td></td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td> GIBCO </td><td> 15140-122 </td><td></td></tr><tr><td>アムホテリシンB </td><td> GIBCO </td><td> 15290-018 </td><td></td></tr><tr><td>定義されたウシ胎児血清（FBS） </td><td> Hyclone社</td><td> SH30070.03 </td><td></td></tr><tr><td> L-アスコルビン酸-2 - リン酸</td><td>シグマ</td><td> A8960-5G </td><td></td></tr><tr><td> L-グルタミン酸</td><td> GIBCO </td><td> 25030-024 </td><td></td></tr><tr><td>デキサメタゾン</td><td>シグマ</td><td> D4902 </td><td></td></tr><tr><td> β-グリセロールリン酸二ナトリウム塩水和物、BioUltra </td><td>シグマ</td><td> G9422-100G </td><td></td></tr><tr><td>リン酸二水素カリウム</td><td>シグマアルドリッチ</td><td> P5655 </td><td></td></tr><tr><td>骨形成アッセイキット</td><td>ミリポア</td><td> PS01802031 </td><td></td></tr><tr><td>マウスIgG2b-PEアイソタイプコントロール</td><td> BDファーミンジェン</td><td> 50808088029 </td><td></td></tr><tr><td> FITC標識マウスIgG2bアイソタイプコントロール</td><td> BDファーミンジェン</td><td> 559532 </td><td></td></tr><tr><td> FITC標識マウスIgG1κアイソタイプ</td><td> BDファーミンジェン</td><td> 11471471 </td><td></td></tr><tr><td> FITC / PEのマウス抗ヒトCD34/CD45 </td><td> BDファーミンジェン</td><td>341071 </td><td></td></tr><tr><td> PEの抗ヒトCD146 </td><td> BDファーミンジェン</td><td> 550315 </td><td></td></tr><tr><td>モノクローナルマウス抗ヒトCD90/FITC </td><td> Daka </td><td> 00034418 </td><td></td></tr><tr><td> PEのマウス抗ヒトCD73 </td><td> BDファーミンジェン</td><td> 550257 </td><td></td></tr><tr><td>抗H CD105/Endoglin PE </td><td> BDファーミンジェン</td><td> FAB10971P </td><td></td></tr><tr><td> PEのマウス抗ヒトCD11b/Mac1 </td><td> BDファーミンジェン</td><td> 5553888 </td><td></td></tr><tr><td> CD44は、PEマウス抗ヒト</td><td> BDファーミンジェン</td><td> 555479 </td><td></td></tr><tr><td>リン酸緩衝液（PBS） </td><td> GIBCO </td><td> 003000 </td><td></td></tr><tr><td> 70μmのセルストレーナー</td><td>ファルコン</td><td> 352360 </td><td></td></tr><tr><td&gt;0.2μmのシリンジフィルター</td><td>マイレクス-GV </td><td> SLGV033RB </td><td></td></tr><tr><td> 25cm 2の培養フラスコ</td><td>シグマアルドリッチ</td><td> Z707481 </td><td></td></tr><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td> BD FACSキャリバー</td><td> BD </td><td> 342975 </td><td></td></tr><tr><td>マイクロプレート分光光度計multiskan </td><td>サーモサイエンティフィック</td><td> 51119200 </td><td></td></tr><tr><td> Fleurcense顕微鏡</td><td>オリンポス</td><td></td><td></td></tr><tr><td>ソフトウェアバージョン2.3.1を流れる</td><td></td><td></td><td></td></tr></tbody></table>

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

<p class="jove_content"開発&gt;親知らずはルーチン矯正治療によって得られることが成人期に幹細胞の容易にアクセス可能なソースです。ヒト歯髄由来幹細胞（hDPSCs）成体幹細胞の通常のソースと比較して多系統分化能力と高い増殖能力を持っている<sup&gt; 1月8日</sup&gt;ので、hDPSCsは、組​​織工学や再生医療における自家移植のための良い候補である可能性があります。これらの利点に加えて、そのようなimmunolodulatory効果などの間葉系幹細胞（MSC）の機能を有するも、同種移植の場合には、hDPSCsがより貴重作る<sup&gt; 6,9,10</sup&gt;。そのため、幹細胞のこのソースを使用する主なステップは、歯髄組織からhDPSCsを単離するための最適なプロトコルを選択することです。この目標を達成するためには、そのような彼らの一般的な表面マーカー＆またそれらの分化能など異なる細胞行動、上の様々な分離条件の影響を調査することが重要です。</p&gt;<p class="jove_content"&gt;このように、ここで我々は影響を受け第三大臼歯歯からヒト歯髄組織を分離し、次にhDPSCsを単離するための文献に基づいて、両方の既存のプロトコルを使用し、<sup&gt; 11月13日</sup&gt;<em&gt;すなわち</em組織外植片から&gt;酵素歯髄組織の解離（DPSC-ED）または伸長（DPSC-OG）。これに関しては、我々は、歯科ダイヤモンドディスクを使用して単離法を容易にしようとしました。次に、これらの細胞はCD146ともSTRO-1のような間質関連マーカー（CD73、CD90、CD105、CD44＆）、造血/内皮細胞マーカー（CD34、CD45及びCD11bを）、血管周囲のマーカーで特徴付け。その後、これらの2つのプロトコルは、定量的ポリメラーゼ連鎖反応（QPCR）＆アリザリンレッド染色の両方による象牙芽細胞への分化能に基づいて比較した。 QPCRは、鉱化関連遺伝子の発現（; ALP、マトリックス細胞外リン糖タンパク質; MEPE＆象牙シアロホスホ·タンパク質; DSPPアルカリホスファターゼ）の評価のために使用された。<sup&gt; 14</sup</p

<ol><li> 酵素的解離（DPSC-ED）により得られたDPSCsは10日、15,18（図1）ここに示されています。分離後約3〜5日に形成するために開始されたコロニーが。 </li><li> 手狭DPSCs（DPSC-OG）は5日、10、13＆18ページの図2に示されています。線維芽細胞様細胞は、平行播種後約5日で注文してフラスコに歯髄組織から移行し始めた。 </li><li> 両方の方法を使用して通路3でDPSCsを図3に示します。DPSCs</...

Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

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

のいずれかを使用して人間の歯髄幹細胞（hDPSCs）の単離および特性を説明する方法<strongパルプ&gt;酵素的解離（DPSC-ED）</strong&gt;または<strong歯髄組織外植片からの幹細胞の&gt;直接伸長（DPSC-OG）。その後に続く</strong&gt;<em&gt; in vitroで</em象牙芽細胞へのhDPSCs両方のタイプの&gt;比較分化。

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 ...

isolation-characterization-comparative-differentiation-human-dental

Research

JoVE Journal

Medicine

26.3K ビュー.  Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. のいずれかを使用して人間の歯髄幹細胞（hDPSCs）の単離および特性を説明する方法酵素的解離（DPSC-ED）または直接伸長（DPSC-OG）。その後に続く in vitroで比較分化。

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

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

分離の特性と人間の歯小嚢細胞のマイクロ Rna を用いた遺伝子組み換え

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

遺伝学

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

がん研究

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

医学

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

離と新鮮な外科的試料からのヒト心室の心筋細胞の機能解析

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

ヒト前立腺癌サンプルからがん幹細胞の単離

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very important to obtain adult stem cells from different tissues such as adipose, umbilical cord, bone marrow and blood. Among the possible sources, dental pulp is particularly interesting because it is easy to obtain in respect of bioethical considerations. Indeed, human Dental Pulp Stem Cells (hDPSCs) are a type of adult stem cells able to differentiate in neuronal-like cells and can be obtained from the third molar of healthy patients (13-19 ages). In particular, the dental pulp was removed with an excavator, cut into small slices, treated with collagenase IV and cultured in a flask. To induce the neuronal differentiation, hDPSCs were stimulated with EGF/bFGF for 2 weeks. Previously, we have demonstrated that during the differentiation process the content of cellular prion Protein (PrPC) in hDPSCs increased. The cytofluorimetric analysis showed an early expression of PrPC that increased after neuronal differentiation process. Ablation of PrPC by siRNA PrP prevented neuronal differentiation induced by EGF/bFGF. In this paper, we illustrate that as we enhanced the isolation, separation and in vitro cultivation methods of hDPSCs with several easy procedures, more efficient cell clones were obtained and large-scale expansion of the mesenchymal stem cells (MSCs) was observed. We also show how the hDPSCs, obtained with methods detailed in the protocol, are an excellent experimental model to study the neuronal differentiation process of MSCs and subsequent cellular and molecular processes.

Here we present a protocol for human Dental Pulp Stem Cells isolation and propagation in order to evaluate the prion protein expression during neuronal differentiation process.

分離、伝達、およびプリオン蛋白質発現ひと歯髄幹細胞の神経分化

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...

発生生物学

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

マウスとヒトの歯からの単一細胞の迅速な分離

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

インビトロ 膵臓系統に向けたヒト歯髄幹細胞の誘導

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

生物工学

Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which are present in both deciduous and permanent teeth, have emerged as a vital stem cell source due to their accessibility, adaptability, and innate differentiation capabilities. DPSCs offer a readily available and abundant reservoir of mesenchymal stem cells, showcasing impressive versatility and potential, particularly for regenerative purposes. Despite their promise, the main hurdle lies in effectively isolating and characterizing DPSCs, given their representation as a minute fraction within dental pulp cells. Equally crucial is the proper preservation of this invaluable cellular resource. The two predominant methods for DPSC isolation are enzymatic digestion (ED) and outgrowth from tissue explants (OG), often referred to as spontaneous growth. This protocol concentrates primarily on the enzymatic digestion approach for DPSC isolation, intricately detailing the steps encompassing extraction, in-lab processing, and cell preservation. Beyond extraction and preservation, the protocol delves into the differentiation prowess of DPSCs. Specifically, it outlines the procedures employed to induce these stem cells to differentiate into adipocytes, osteoblasts, and chondrocytes, showcasing their multipotent attributes. Subsequent utilization of colorimetric staining techniques facilitates accurate visualization and confirmation of successful differentiation, thereby validating the caliber and functionality of the isolated DPSCs. This comprehensive protocol functions as a blueprint encompassing the entire spectrum of dental pulp stem cell extraction, cultivation, preservation, and characterization. It underscores the substantial potential harbored by DPSCs, propelling forward stem cell exploration and holding promise for future regenerative and therapeutic breakthroughs.

This protocol emphasizes the extraction, culture, and preservation of multipotent stem cells from dental pulp through enzymatic digestion. Additionally, it demonstrates their potential to differentiate into osteoblasts, adipocytes, and chondrocytes, highlighting the importance of precision and consistency in the process.

ヒト落葉歯および永久歯からの歯髄幹細胞の単離、培養、および特性評価

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which ...

歯髄幹細胞の初代培養の確立

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

著者スポットライト:歯髄幹細胞による組織再生と疾患モデリングの進歩

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

歯髄幹細胞の初代培養

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...

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

要約

さらに動画を探す

この動画の章

分離の特性と人間の歯小嚢細胞のマイクロ Rna を用いた遺伝子組み換え

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

CD133の患者由来の細胞培養および分離

離と新鮮な外科的試料からのヒト心室の心筋細胞の機能解析

ヒト前立腺癌サンプルからがん幹細胞の単離

分離、伝達、およびプリオン蛋白質発現ひと歯髄幹細胞の神経分化

マウスとヒトの歯からの単一細胞の迅速な分離

インビトロ膵臓系統に向けたヒト歯髄幹細胞の誘導

ヒト落葉歯および永久歯からの歯髄幹細胞の単離、培養、および特性評価

歯髄幹細胞の初代培養

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

要約

さらに動画を探す

この動画の章

分離の特性と人間の歯小嚢細胞のマイクロ Rna を用いた遺伝子組み換え

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

CD133の患者由来の細胞培養および分離

離と新鮮な外科的試料からのヒト心室の心筋細胞の機能解析

ヒト前立腺癌サンプルからがん幹細胞の単離

分離、伝達、およびプリオン蛋白質発現ひと歯髄幹細胞の神経分化

マウスとヒトの歯からの単一細胞の迅速な分離

インビトロ 膵臓系統に向けたヒト歯髄幹細胞の誘導

ヒト落葉歯および永久歯からの歯髄幹細胞の単離、培養、および特性評価

歯髄幹細胞の初代培養

インビトロ膵臓系統に向けたヒト歯髄幹細胞の誘導