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>
	<li>Volponi, A. A., Pang, Y., Sharpe, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+biological+tooth+repair+and+regeneration.">Stem cell-based biological tooth repair and regeneration.</a> Trends Cell Biol. 20, 715-722 (2010).</li><li>Nosrat, I. V., Widenfalk, J., Olson, L., Nosrat, C. A. <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+cells+produce+neurotrophic+factors,+interact+with+trigeminal+neurons+in+vitro,+and+rescue+motoneurons+after+spinal+cord+injury.">Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury.</a> Dev. Biol. 238, 120-132 (2001).</li><li>Gandia, 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=Human+dental+pulp+stem+cells+improve+left+ventricular+function,+induce+angiogenesis,+and+reduce+infarct+size+in+rats+with+acute+myocardial+infarction.">Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction.</a> Stem Cells. 26, 638-645 (2008).</li><li>Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+human+dental+pulp+stem+cells+(DPSCs)+in+vitro+and+in+vivo.">Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.</a> Proc. Natl. Acad. Sci. U. S. A.. 97, 13625-13630 (2000).</li><li>Graziano, A., d'Aquino, R., Laino, G., Papaccio, G. <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:+a+promising+tool+for+bone+regeneration.">Dental pulp stem cells: a promising tool for bone regeneration.</a> Stem Cell Rev. 4, 21-26 (2008).</li><li>Kerkis, I., 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=Early+transplantation+of+human+immature+dental+pulp+stem+cells+from+baby+teeth+to+golden+retriever+muscular+dystrophy+(GRMD)+dogs:+Local+or+systemic.">Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic.</a> J. Transl. Med. 6, 35 (2008).</li><li>Onyekwelu, O., Seppala, M., Zoupa, M., Cobourne, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+development:+2.+Regenerating+teeth+in+the+laboratory.">Tooth development: 2. Regenerating teeth in the laboratory.</a> Dent. Update. 34, 20-29 (2007).</li><li>Cordeiro, M. 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=Dental+pulp+tissue+engineering+with+stem+cells+from+exfoliated+deciduous+teeth.">Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth.</a> J. Endod. 34 (08), 962-969 (2008).</li><li>Pierdomenico, L., 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=Multipotent+mesenchymal+stem+cells+with+immunosuppressive+activity+can+be+easily+isolated+from+dental+pulp.">Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp.</a> Transplantation. 80, 836-842 (2005).</li><li>de Mendonca Costa, 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=Reconstruction+of+large+cranial+defects+in+nonimmunosuppressed+experimental+design+with+human+dental+pulp+stem+cells.">Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells.</a> J. Craniofac. Surg. 19, 204-210 (2008).</li><li>Tirino, V., 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=Methods+for+the+identification,+characterization+and+banking+of+human+DPSCs:+current+strategies+and+perspectives.">Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives.</a> Stem Cell Rev. 7, 608-615 (2011).</li><li>Tirino, V., Paino, F., De Rosa, A., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation,+characterization,+and+banking+of+human+dental+pulp+stem+cells.">Identification, isolation, characterization, and banking of human dental pulp stem cells.</a> Methods Mol. Biol. 879, 443-463 (2012).</li><li>Eslaminejad, M. B., Vahabi, S., Shariati, M., Nazarian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Growth+and+Characterization+of+Stem+Cells+from+Human+Dental+Pulp+of+Deciduous+Versus+Permanent+Teeth.">In vitro Growth and Characterization of Stem Cells from Human Dental Pulp of Deciduous Versus Permanent Teeth.</a> J. Dent. (Tehran). 7, 185-195 (2010).</li><li>Wei, X., Ling, J., Wu, L., Liu, L., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mineralization+markers+in+dental+pulp+cells.">Expression of mineralization markers in dental pulp cells.</a> J. Endod. 33, 703-708 (2007).</li><li>Nombela-Arrieta, C., Ritz, J., Silberstein, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elusive+nature+and+function+of+mesenchymal+stem+cells.">The elusive nature and function of mesenchymal stem cells.</a> Nat. Rev. Mol Cell Biol. 12, 126-131 (2011).</li><li>Huang, G. T., Gronthos, S., Shi, S. <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+dental+tissues+vs.+those+from+other+sources:+their+biology+and+role+in+regenerative+medicine.">Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine.</a> J. Dent. Res. 88, 792-806 (2009).</li><li>Graziano, 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=Scaffold's+surface+geometry+significantly+affects+human+stem+cell+bone+tissue+engineering.">Scaffold's surface geometry significantly affects human stem cell bone tissue engineering.</a> J. Cell Physiol. 214, 166-172 (2008).</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+dental+pulp+stem+cells:+from+biology+to+clinical+applications.">Human dental pulp stem cells: from biology to clinical applications.</a> J. Exp. Zool. B. Mol. Dev. Evol. 312, 408-415 (2009).</li><li>Mitsiadis, T. A., Feki, A., Papaccio, G., Caton, J. <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,+niches,+and+notch+signaling+in+tooth+injury.">Dental pulp stem cells, niches, and notch signaling in tooth injury.</a> Adv. Dent. Res. 23, 275-279 (2011).</li><li>Shi, S., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+niche+of+postnatal+mesenchymal+stem+cells+in+human+bone+marrow+and+dental+pulp.">Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp.</a> J. Bone Miner. Res. 18, 696-704 (2003).</li><li>Tomic, 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=Immunomodulatory+properties+of+mesenchymal+stem+cells+derived+from+dental+pulp+and+dental+follicle+are+susceptible+to+activation+by+toll-like+receptor+agonists.">Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.</a> Stem Cells Dev. 20, 695-708 (2011).</li><li>Tsukamoto, 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=Mineralized+nodule+formation+by+cultures+of+human+dental+pulp-derived+fibroblasts.">Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.</a> Arch. Oral Biol. 37, 1045-1055 (1992).</li><li>About, I., 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+dentin+production+in+vitro.">Human dentin production in vitro.</a> Exp. Cell Res. 258, 33-41 (2000).</li><li>Couble, M. L., 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=Odontoblast+differentiation+of+human+dental+pulp+cells+in+explant+cultures.">Odontoblast differentiation of human dental pulp cells in explant cultures.</a> Calcif. Tissue Int. 66, 129-138 (2000).</li><li>Onishi, T., Kinoshita, S., Shintani, S., Sobue, S., Ooshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+proliferation+and+differentiation+of+dog+dental+pulp+cells+in+serum-free+culture+medium+by+insulin-like+growth+factor.">Stimulation of proliferation and differentiation of dog dental pulp cells in serum-free culture medium by insulin-like growth factor.</a> Arch. Oral Biol. 44 (99), 361-371 (1999).</li><li>Huang, G. T., Sonoyama, W., Chen, J., Park, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+human+dental+pulp+cells:+various+isolation+methods+and+culturing+environments.">In vitro characterization of human dental pulp cells: various isolation methods and culturing environments.</a> Cell Tissue Res. 324, 225-236 (2006).</li><li>Bakopoulou, 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=Assessment+of+the+impact+of+two+different+isolation+methods+on+the+osteo/odontogenic+differentiation+potential+of+human+dental+stem+cells+derived+from+deciduous+teeth.">Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.</a> Calcif. Tissue Int. 88, 130-141 (2011).</li><li>Curtis, K. 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>

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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

26.2K 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

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 ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

子宮摘出標本から初代ヒト子宮内膜間質細胞の確立するための2つの方法

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

ミニブタの滑液および骨髄由来の間葉系幹細胞の単離および細胞表現型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

フローサイトメトリーを用いたひと脂肪組織での免疫細胞のキャラクタリゼーション

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

歯科用インプラント ・異種材料の口腔バイオ フィルム形成

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

ヒト脂肪由来間葉系幹細胞の単離・増殖のための酵素フリー法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

ひと臍帯血由来 cd34 陽性造血幹細胞の体外増幅+細胞を用いたバルプロ酸

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

異なる均質化法を通じてマウス脳/脊髄から取り出された複数の細胞型の同時フロー細胞特性評価

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...