3R01AR054458 - 02S2：この作品は、関節炎、筋骨格系の国立研究所と皮膚疾患からの助成金によって支えられて。

1。日1 1）細胞をプレート<ol><li>少なくとも18-24時間前にラベリング〜80％のコンフルエントでT75フラスコでプレートのhMSCを。代替船のための命令については、表1を参照してください。 </li></ol> 2。日2 2）標識溶液を準備します。この準備は、400μgの鉄/ mlの濃度で80％コンフルエントに一つ（1）T75フラスコにラベルを付けます。代替船のための命令については、表1を参照してください。 <ol><li> 15 mlコニカルチューブに：無血清培地1ml及びferumoxytolの93.1μL（30 mg / mlストック）を混合して溶液（溶液1）を作成します。 </li><li>第二15 mlコニカルチューブに：無血清培地1ml及び硫酸プロタミンの7μlの（10 mg / mlストック）を混合することにより、第二の溶液（溶液2）を作成します。 </li><li>各ソリューションは、5分間休憩することができます。 </li><li>一緒に解決策1と2を追加。ゆっくりと新しい標識溶液を混ぜる。ソリューションは、USPIO - PROを許可するために5分間休息することができますtamine硫酸複合体が形成する。 </li><li> SPIO / 7 mlの最終ボリュームのプロタミン硫酸塩溶液との混合2mlに無血清培地5 mlを加える。 </li></ol> 3）標識のための細胞を準備する<ol><li>細胞から培地を吸引除去する。 </li><li>静かに造影剤の取り込みと標識効率を損なう可能性の残余血清タンパク質およびまたはその他のメディアコンポーネントを洗うために予め温めておいたのMg / CaのフリーD - PBSまたは無血清培地の2〜3 mlの細胞を1回洗浄する。リンス液を吸引除去する。 </li></ol> 4）ラベルの細胞<ol><li>細胞へのソリューションをラベリングの完全な7ミリリットルを追加。 </li><li>場所は、インキュベーターにセル（37℃/ 5％CO 2）と細胞は4時間のラベリング溶液中でインキュベートすることができます。 </li><li> 10％の最終的な血清濃度を達成するために細胞のラベリングソリューションにFCS 700μlを添加する。血清は、細胞が慣れているから豊かな環境を再確立するために、そしてマイルを支援するために追加されます。無血清環境に24時間曝露に急激な変化から生じる可能性が細胞死をnimizing。 </li><li>細胞はさらに20時​​間のラベリングソリューションをインキュベートすることができます。 </li></ol> 3。日3 5）標識細胞を準備します。 <ol><li>細胞からの標識溶液を吸引除去する。 </li><li>静かに予め温めておいたのMg / CaのフリーD - PBSの2-3 mlの細胞をすすいでください。 PBSを吸引除去する。それは、MgおよびCaは、次の手順でトリプシンの効率が低下する傾向があるとしてMg / CaのフリーPBSを使用することが重要です。 </li><li>細胞に予め温めておいた0.05％トリプシン液2 mlを追加し、フラスコの表面全体を確実にするために前後にフラスコを傾けはトリプシンの薄層で覆われている。インキュベーター（37℃/ 5％CO 2）内のセルを配置し、細胞がプレートからデタッチし始めるまで細胞が5-7分間、または休息することができます。それは、顕微鏡下で剥離を確認する必要があるかもしれません。 FAに必要に応じてゆっくりとフラスコの側面をタップ細胞表面の剥離をcilitate。 </li><li>トリプシンを中和するためにフラスコに予め温めておいた完全培地4mlを追加します。ゆっくりと上下に数回をトリプシン/メディア/電池ソリューションをピペッティングすることによりフラスコを洗う。 15 mlコニカルチューブ中で、ソリューション全体を収集する。 5分間で400 RCFにおける細胞溶液を遠心してください。 </li><li>細胞ペレットを乱すことなく上清を注意深く吸引除去する。メディア5mlの（を含む血清または無血清）で細胞を再懸濁します。 5分間で400 RCFにおける細胞溶液を遠心してください。 </li><li>ステップ5.5で説明したセルの洗浄を繰り返します。合計では、細胞を培地にし、細胞の表面上に残留、無料の造影剤を除去するために3回リンスする。 </li><li>細胞は、前洗浄中に失われるので、最も正確な細胞数を達成するために、この時点で細胞を数える。細胞の生存率のテストを実行します。セルは、その後の分析とや実験的なアプリケーションのための準備が整いました。 MRイメージングは​​、T1 - wで実行することができますし、<html> T2 - wのパラメータは、ferumoxytolはT2 - W造影剤であるがため、ferumoxytolはまた、T1効果を発揮する。画像ferumoxytol標識された細胞に使用できる代表的なT2 - W MRIパラメータが含まれています：FSEまたは2000から2500ミリ秒60〜80ミリ秒のTEのTRとSE_Multisliceシーケンスを。 T1 - Wの画像を取得するには、FSEまたはSE_MultisliceシーケンスのTEの値を下げるか、または高いTRと低TEでのGREシーケンスを使用してください。図4は、標識幹細胞イメージングのためのin vivoでのアプリケーションの可能性を示しています。 </li></ol> 4。代表的な結果： 標識された細胞は、T2強調画像で暗くなる重要なまたは負コントラストの効果とT1強調MR画像（図2）上で明るくまたは正のコントラスト効果を示しています。縦MRイメージングと実行可能性のテストは5日間実行されるポストラベリングは、有意なMR信号と未標識コントロール（データは示さず）に比べて生存率に有意な影響は示されなかった。 jove_content&quot;&gt;標識幹細胞は、その後、種々の細胞型に分化させるまたは静脈内in vivoでの調査でのために注入することができます。 <img alt="図1" src="/files/ftp_upload/3482/3482fig1.jpg" /> 図1酸化鉄ナノ粒子、ferumoxytolで幹細胞を標識するための回路図のタイムライン。 <img alt="図2" src="/files/ftp_upload/3482/3482fig2.jpg" /> 図2。細胞ペレット中の細胞と矢状断面thrroughエッペンドルフチューブのMR画像。 A）非標識のコントロールが。 B）標識された細胞は、T2強調FSEまたはSE_MultisliceイメージングとT1強調GREシーケンスのT1または正の造影剤の効果に重大なT2または負の造影剤の効果を発揮。 <img alt="図3" src="/files/ftp_upload/3482/3482fig3.jpg" /> 図3。細胞のPELの細胞とのエッペンドルフチューブの矢状断面ことができます。わずか1万人のferumoxytol標識された細胞は、T2 - W MRイメージングを介して検出することができます。 <img alt="図4" src="/files/ftp_upload/3482/3482fig4.jpg" /> 図4。ネズミの脳室に注入ferumoxytol標識幹細胞のMRの可視化。 A）軸、USPIO標識幹細胞の注入なしでマウスバリンのMR画像。 B）冠状軸、C）、およびD）矢状MR画像は、マウス脳内に注入されたUSPIOで標識された幹細胞（白矢印）のT2、否定的なコントラストの効果を描いた。 <img alt="表1" src="/files/ftp_upload/3482/3482table1.jpg" /> 別の容器に細胞を標識する表1。数量adaptions。

<ol>
	<li>Narsinh, K. H., Plews, J., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+induced+pluripotent+and+embryonic+stem+cells:+fraternal+or+identical+twins?.">Comparison of human induced pluripotent and embryonic stem cells: fraternal or identical twins?.</a> Mol Ther. 19, 635-638 (2011).</li><li>Bulte, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MRI+cell+tracking:+clinical+studies.">In vivo MRI cell tracking: clinical studies.</a> AJR. Am. J. Roentgenol. 193, 314-325 (2009).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+Iron+Oxide+Nanoparticles+for+Non-Invasive+in+vivo+Tracking+with+MR+Imaging.">Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging.</a> J. Vis. Exp. (13), e685-e685 (2008).</li><li>Tallheden, T., Nannmark, U., Lorentzon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+imaging+of+magnetically+labeled+human+embryonic+stem+cells.">In vivo MR imaging of magnetically labeled human embryonic stem cells.</a> Life. Sci. 79, 999-1006 (2006).</li><li>Jung, C. W., Jacobs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+properties+of+superparamagnetic+iron+oxide+MR+contrast+agents:+ferumoxides,+ferumoxtran,+ferumoxsil.">Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil.</a> Magn. Reson. Imaging. 13, 661-674 (1995).</li><li>Coyne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferumoxytol+for+treatment+of+iron+deficiency+anemia+in+patients+with+chronic+kidney+disease.">Ferumoxytol for treatment of iron deficiency anemia in patients with chronic kidney disease.</a> Expert. Opin. Pharmacother. 10, 2563-2568 (2009).</li><li>Li, Z., Suzuki, Y., Huang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+reporter+gene+and+iron+particle+labeling+for+tracking+fate+of+human+embryonic+stem+cells+and+differentiated+endothelial+cells+in+living+subjects.">Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects.</a> Stem Cells. 26, 864-873 (2008).</li><li>Metz, S., Bonaterra, G., Rudelius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+human+monocytes+to+phagocytose+approved+iron+oxide+MR+contrast+agents+in+vitro.">Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro.</a> Eur. Radiol. 14, 1851-1858 (2004).</li><li>Nedopil, A., Klenk, C., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+signal+characteristics+of+viable+and+apoptotic+human+mesenchymal+stem+cells+in+matrix-associated+stem+cell+implants+for+treatment+of+osteoarthritis.">MR signal characteristics of viable and apoptotic human mesenchymal stem cells in matrix-associated stem cell implants for treatment of osteoarthritis.</a> Invest. Radiol. 45, 634-640 (2010).</li><li>Kraitchman, D. L., Heldman, A. W., Atalar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+magnetic+resonance+imaging+of+mesenchymal+stem+cells+in+myocardial+infarction.">In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction.</a> Circulation. 107, 2290-2293 (2003).</li><li>Stuckey, D. J., Carr, C. A., Martin-Rendon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+particles+for+noninvasive+monitoring+of+bone+marrow+stromal+cell+engraftment+into,+and+isolation+of+viable+engrafted+donor+cells+from,+the+heart.">Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.</a> Stem Cells. 24, 1968-1975 (2006).</li><li>Henning, T. D., Sutton, E. J., Kim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+ferucarbotran+on+the+chondrogenesis+of+human+mesenchymal+stem+cells.">The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells.</a> Contrast. Media. Mol. Imaging. 4, 165-173 (2009).</li><li>Arbab, A. S., Yocum, G. T., Kalish, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Nedopil, A. J., Mandrussow, L. G., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+Ferumoxides+Labeled+Human+Mesenchymal+Stem+Cells+in+Cartilage+Defects.">Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects.</a> J. Vis. Exp. (38), e1793-e1793 (2010).</li><li>Arbab, A. S., Yocum, G. T., Wilson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transfection+agents+in+forming+complexes+with+ferumoxides,+cell+labeling+efficiency,+and+cellular+viability.">Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability.</a> Mol Imaging. 3, 24-32 (2004).</li><li>Babic, M., Horak, D., Trchova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(L-lysine)-modified+iron+oxide+nanoparticles+for+stem+cell+labeling.">Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.</a> Bioconjug Chem. 19, 740-750 (2008).</li><li>Golovko, D. M., T, H. e. n. n. i. n. g., Bauer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+stem+cell+labeling+with+ferucarbotran+and+protamine.">Accelerated stem cell labeling with ferucarbotran and protamine.</a> Eur. Radiol. 20, 640-648 (2010).</li><li>Lu, M., Cohen, M. H., Rieves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+report:+Ferumoxytol+for+intravenous+iron+therapy+in+adult+patients+with+chronic+kidney+disease.">FDA report: Ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease.</a> Am. J. Hematol. 85, 315-319 (2010).</li></ol>

この研究への全貢献者には開示を提供していません。

幹細胞のengraftmentsの有効性を改善することは再生医療の進歩にとって重要です。 in vivoで幹細胞の非侵襲可視化技術は大幅に生着の成功の成果につながるメカニズムを理解する当社の能力を高める。このような我々が実証された手順としてMRの可視化のための磁気標識は、MRイメージングと幹細胞の in vivo追跡にできます。磁気標識された幹細胞は、以前に標的組織に移植?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td> D - MEM高グルコース</td><td>シグマ</td><td> D5648 </td><td>使用されるべき所望の幹細胞株、あるいは他の基礎培地</td></tr><tr><td> D - PBS（Ca + +の 、Mg + +のフリー） </td><td> GIBCO </td><td> 14190-144 </td><td></td></tr><tr><td>トリプシン- EDTA 0.05パーセント</td><td>インビトロジェン</td><td> 25300-120 </td><td></td></tr><tr><td>ウシ胎児血清（FBS） </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td> Ferumoxytol （Feraheme） </td><td> AMAG </td><td> 59338-0775-​​01 </td><td></td></tr><tr><td>硫酸プロタミン</td><td> APP薬学。 </td><td> 22930 </td><td></td></tr>
</tbody>
</table>

labeling stem cells with ferumoxytol, an fda-approved iron oxide nanoparticle

幹細胞ベースの治療は再生医療の分野のための大きな潜在的可能性を提供します。しかし、多くの移植細胞の生体内動態に関する理解されていない。繰り返しin vivoで移植された幹細胞を監視する非侵襲的方法は、研究者が直接幹細胞移植を監視し、成功または失敗した移植の成果を識別できるようになります。 幹細胞の広い範囲は、無数のアプリケーションのために検討され続けている。ヒト胚性腎臓293（HEK293）細胞、ヒト間葉系幹細胞（hMSCの）と人工多能性幹（iPS）細胞：このプロトコルは、3種類の幹細胞集団に焦点を当てています。 HEK 293細胞は、剪断されたアデノウイルス5 DNAとの文化の中で増殖したヒト胚性腎細胞から派生しています。それらが容易に培養されているため、これらの細胞が広く研究に使用されている、急速に増大し、容易にトランスフェクトされています。 hMSCsは、成人の骨髄に見出される。これらの細胞は約多能または細胞の運命の限られた数に分化する能力を維持しながら、n個の未分化細胞として複製される。 hMSCsは、骨芽細胞、脂肪細胞、軟骨、腱、筋肉、及び骨髄間質を含む間葉系組織、の系統に分化することができる。 iPS細胞は、遺伝的遺伝子と胚性幹細胞の定義するプロパティと同様の因子を発現するように変更されている成人の細胞を再プログラムしている。これらの細胞は、彼らがすべての細胞系譜1に分化する能力を持つ多能性の意味です。 hMSCsとiPS細胞の両方は、生体内で組織再生能力を実証している。 一緒に超常磁性酸化鉄（SPIO）ナノ粒子の細胞のラベルの使用による磁気共鳴（MR）イメージングが近くに微細な解剖学的に解決し、幹細胞の生体内追跡のために効果的な証明されている、それより長い血中半減期は、長手方向のイメージングおよび許可する高sensitivitSPIOのMRイメージングで提供される細胞の検出のためのyは2-4ナノ粒子。さらに、SPIOsの使用とMRイメージングは​​臨床的に変換可能です。 SPIOsはデキストラン、carboxydextranまたは血漿成分からbioreactive鉄のコアを含むように機能する澱粉の表面コートと酸化鉄のコアで構成されています。これらのエージェントは、T2強調MR画像5上の減少信号につながる局所磁場の不均一性を作成します。残念ながら、SPIOsはもはや製造されています。第二世代、超小型SPIOs（USPIO）が、しかし、実行可能な代案を提供します。 Ferumoxytol（FerahemeTM）はポリグルコース、ソルビトールcarboxymethyletherコートに囲まれた非化学量論的マグネタイトのコアからなる1 USPIOです。光散乱によって決定されるferumoxytolのコロイド、粒子サイズは17〜30 nmである。分子量は750 kDaのであり、そして2T MRI分野で一定の緩和度は58.609 MM - 1秒-1強度4です。 Ferumoxytolは最近FDA -承認された腎不全6患者における鉄欠乏の治療のためのsは、鉄のサプリメント。当社グループは、細胞標識のアプリケーションのための&quot;オフラベル&quot;使用されてこのエージェントを適用しています。私たちの技術は、MR画像上で標識された細胞の重要なMR信号の効果につながるferumoxytol持つ幹細胞の効率的なラベリングを示しています。この手法は、前臨床および臨床設定における幹細胞療法の非侵襲的なモニタリングに適用されることがあります。

Watch this Scientific Journal Video about Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle at JoVE.com

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

我々は、FDAに承認された、超常磁性酸化鉄（SPIO）、ferumoxytol（Feraheme）で幹細胞を標識して追跡するためのテクニックを説明します。可視化のために磁気共鳴（MR）イメージングを利用したこの細胞のイメージング技術は、患者の成功または失敗した幹細胞のengraftmentsの長期的な監視と診断のための容易にアクセス可能です。

Ferumoxytol、FDA承認酸化鉄ナノ粒子で幹細胞のラベリング

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

labeling-stem-cells-with-ferumoxytol-an-fda-approved-iron-oxide

Research

JoVE Journal

Medicine

20.7K ビュー.  Molecular Imaging Program at Stanford (MIPS). 我々は、FDAに承認された、超常磁性酸化鉄（SPIO）、ferumoxytol（Feraheme）で幹細胞を標識して追跡するためのテクニックを説明します。可視化のために磁気共鳴（MR）イメージングを利用したこの細胞のイメージング技術は、患者の成功または失敗した幹細胞のengraftmentsの長期的な監視と診断のための容易にアクセス可能です。

ビデオ: Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

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 ...

医学

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

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 ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

フォルスコリン誘発性腸オルガノイドに腫れ：<em&gt;インビトロ</em&gt;アッセイ嚢胞性線維症患者における薬物反応を評価するための

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

鎌状赤血球症の患者のための連続的なマニュアル交換輸血：鉄過負荷を回避するための効率的な方法

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

超常磁性鉄酸化物ナノプローブの合成、特性解析、応用による肺外結核検出

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

小さい干渉の RNA のマクロファージへの配信のための車両としてポリエチレンイミン被覆された酸化鉄ナノ粒子の in VitroとIn Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

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. ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

外傷性脳損傷マウスモデルにおける鼻腔内送達後のMRIを用いた超常磁性酸化鉄標識間葉系幹細胞の追跡

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

カルシウム蛍光色素標識とナノ粒子追跡解析を用いたヒトの尿中ナノ結晶の推定

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...