この作品は、OTKA（ハンガリー科学研究費）D45933、T049621、TET（ハンガリー科学技術振興財団）A4/04、Arg-17/2006とSIN、Bolyai、ÖvegesのフェローシップとTÁMOPによってサポートされていました4.2.2-08 / 1 / KMR - 2008 - 0004および4.2.1 / B 09/1/KMR-2010-0001。 OTKA 83803。我々は、Voice overを提供するためにウィリアムのGesztesに感謝します。

1。 H9c2 cardiomyoblast細胞の準備 H9c2ラットcardiomyoblastsは、ATCC（ヴェーゼル、ドイツ）から入手し、高グルコース（4.5 g / L）のDMEM、10％ウシ胎児血清、4mMのL -グルタミン、100 U / mlペニシリンおよび100μg/ mlのストレプトマイシンを含むで増殖させた。 H9c2筋芽細胞株は、ラット胚の心臓に由来する、それが骨格と心筋8,9の両方のin vitroモデルとして使用されます。 H9c2細胞の12ウェルプレートを準備します。 <ol><li> H9c2細胞のペトリ皿から培地を削除します。 </li><li> 2〜3ミリリットル0.05％トリプシン/ EDTAを5mlのPBSとダイジェストで細胞を2回洗浄する。 </li><li> 37℃インキュベーター内で5分間、ペトリ皿を置きます。 </li><li> 10ミリリットルのDMEM培地とトリプシンを阻害する。 </li><li> 8分間1200rpmで細胞をスピンダウン。 </li><li> 1ミリリットルDMEM培地とトリパンブルー染色を用いて血球計算板でカウント細胞上清、再懸濁し、細胞を除去した後。</li><li> 12ウェルプレートにウェル当たり2ミリリットルDMEMでシード3万H9c2細胞を。 </li><li> 24時間37℃のCO 2インキュベーター内にプレートを置きます。 </li></ol> 2。シミュレートされた虚血/再灌流虚血/再灌流はH9c2細胞培養で酸素グルコース欠乏（OGD）を実行することにより、in vitroでシミュレートされました。この手順は、OGD中の細胞の観察を可能にPeCon細胞培養系（Erbach、ドイツ）とツァイス共焦点顕微鏡のステージで行われました。 <ol><li> H9c2細胞の12ウェルプレートから吸引して培養液を除去。 </li><li> PBSで2回細胞を洗浄する。 </li><li>ウェルあたり3 mlのグルコースフリー培地を追加。 </li><li>細胞の培養系にプレートを置きます。 </li><li>インキュベーションシステムのうち、酸素をパージするために窒素ガスを開始します。 </li><li>その後、タイマーを開始する酸素のレベルが0.5％に達するまで待ちます。 0.5％の酸素が約3 mmHgの部分的な緊張を意味します。 1〜対象セルシミュレートされた虚血の50分。 </li><li>シミュレートされた虚血の終わりには、細胞からグルコース培地を取り除く。 </li><li>ウェルにピペット2ミリリットル通常の培養培地。 </li><li> 37℃CO 2インキュベーターで30分間、12ウェルプレートを置きますが、これは&quot;再灌流の期間&quot;です。 </li></ol> 3。準備救助間葉系幹細胞ヒト由来の骨髄は、4 mM L -グルタミン、T75フラスコに入れ、10％ウシ胎児血清（FCS）、100 U / mlペニシリンおよび100μg/ mlのストレプトマイシンを含むダルベッコ変法イーグル培地（DMEM）培地で希釈し、 1g /リットルのグルコース。フラスコは、3日間、5％CO 2の完全な加湿雰囲気中で37℃でインキュベートした。潜伏期間の後にフラスコや骨髄の残りのコンポーネントの表面に付着したBMSCを、PBSで洗浄することにより排除された。使用されるBMSCを、実験1と5の通路の間であった。 IDEN細胞のティティは、フローサイトメトリーによる系統特異的細胞表面マーカーの存在によって確認された。造血LINAGE -特異的表面マーカー（CD34、CD45）と間葉の表面マーカー（CD73、CD90、CD105およびCD166）を調べた。 シミュレートされた虚血時には、移植用間葉系幹細胞を救う準備。 <ol><li> VybrantはDMEM培地で1:200の割合で、DID蛍光色素を希釈する。 </li><li>救助の細胞から培地を吸引除去し、Vybrantがやったと300μlのDMEMを加える。 </li><li> 37℃CO 2インキュベーターで30分間場所ペトリ皿。 </li><li>間葉系幹細胞のペトリ皿から培地を削除します。 </li><li> 2〜3ミリリットル0.05％トリプシン/ EDTAを5mlのPBSとダイジェストで細胞を2回洗浄する。 </li><li> 37℃インキュベーター内で5分間、ペトリ皿を置きます。 </li><li> 10ミリリットルのDMEM培地とトリプシンを阻害する。 </li><li> 8分間1200rpmで細胞をスピンダウン。 </li><li>スーパを除去した後1ミリリットルDMEM培地およびトリパンブルー染色を用いて血球計算板でカウント細胞におけるrnatant、再懸細胞。 </li><li>再灌流の最後の30分後に虚血後のH9c2心筋芽細胞にも当たり20,000の間葉系幹細胞を加える。 </li><li> 24時間37℃CO 2インキュベーターに場所細胞の共培養。 </li></ol> 4。共焦点顕微鏡による解析シミュレートされた虚血/再灌流障害の後、細胞を24時間、通常の培養培地中でインキュベートされる。 <ol><li> 24時間は、PBSで細胞を2回洗浄した後。 </li><li> 30分間、500μlのLIVE / DEAD色素溶液（5 nMのカルセインと400nmのPBSでエチジウム-ホモ二量体-2）（5 × 10 4細胞/ ml）で細胞を染色。 </li></ol>形態と蛍光によって選択された生細胞と死細胞のための共焦点画像の評価は、ImageJのソフトウェア（国立衛生研究所、米国）で行うことができます。 cの場合にO -文化、MSCはそのVybrantのDID細胞標識のためにH9c2細胞と区別することができます。死細胞の割合は、盲検10の各カルチャのビューの4つの独立した分野（10倍目標）で評価することができます。 42 mmのガラス製カバースリップ上で培養H9c2 cardiomyoblastsはまた共焦点顕微鏡の細胞の培養系を用いて虚血/再灌流障害中および後の時間経過ビデオ顕微鏡で調べることができる。このような細胞融合と細胞間のナノチューブの形成などの細胞間相互作用は、時間をかけて研究することができる。 5。フローサイトメトリーによる解析シミュレートされた虚血/再灌流障害の後、細胞を24時間、通常の培養培地中でインキュベートされる。 <ol><li> 24時間後、培地を収穫。いくつかの死んだ細胞が24時間のインキュベーション中に、培養皿の表面から剥離している可能性があるため、また、培地を収集することは非常に重要です。る。 </li><li> 0.05％トリプシン- EDTAを含むPBSとダイジェストで細胞を2回洗浄する。 </li><li> DMEM培地とトリプシンを阻害する。 </li><li> 8分、1200 RPM（約150〜200グラム）で、細胞と収穫された培養液をスピンダウン。 </li><li>上清を捨てる。 </li><li> 30分間500μLデッド/ライブ色素溶液（5 nMのカルセインと400nmのPBSでエチジウム-ホモ二量体-2）（5 × 10 4細胞/ ml）で細胞を懸濁する。 </li><li>フローサイトメトリーチューブに細胞を移す。 </li><li> CellQuest Proソフトウェアを起動します。 </li><li>測定のためのフローサイトメーターを設定するには、生細胞と死細胞のコントロールサンプルを準備する。 <ol class="lalpha"><li>ポイント1で説明したように12ウェルプレートに3 × 4の密度でプレートH9c2細胞。 </li><li>ポイント1で説明したように、単一のトリプシン処理で細胞のコントロールを生きて準備。 </li><li> 1時間に10μMH 2 O 2で処理することにより、死細胞のコントロールを準備します。 </li><li>トリプシン処理後、resuspen500μlのLIVE / DEAD染料溶液（5 nMのカルセイン- AMおよび400nmのPBSでエチジウム-ホモ二量体A - 2）のd生と死細胞のコントロール。 </li></ol></li><li>死んだ細胞は正のカルセイン負とエチジウムホモ二量体である間、生きている細胞は、負のカルセイン正とエチジウムホモ二量体になるように機器設定を調整する必要があります。 </li><li>グラフと統計分析上のバーが、これらの値に対して実行できるようにこれらの人口の割合を表現することができる。 </li><li>別の実験群を測定する。 </li></ol> 6。代表的な結果： 我々の結果は、追加された間葉系幹細胞は虚血後の心筋細胞の生存率を改善できることを示した。共焦点顕微鏡画像は、おそらく観察保護10で重要な役割を果たしているような部分的な膜の接続や細胞間のナノチューブのような直接的な細胞間相互作用を、明らかになった。これらのナノチューブは、いくつかのセルdの距離にまたがっている場合iameters、およびそれらの直径は200〜500 nmの（白い矢印）の間であった。 <img src="/files/ftp_upload/3575/3575fig1.jpg" alt="図1" /> 図1。セル間のナノチューブ形成。ナノチューブの形成（白矢印）はVybrant DIOラベル付けcardiomyoblastsとVybrant DIDのラベルが付いた葉系幹細胞との間で観察された。 フローサイトメトリー分析は、生存と壊死cardiomyoblastsと間​​葉系幹細胞の集団を示した。興味深いことに、我々はまた、カルセインが含まれている第三に、&quot;負傷&quot;cardiomyoblastの細胞集団を観察しただけでなく、死んだ細胞、エチジウムホモ二量体（図2）の指標が含まれていました。 <img src="/files/ftp_upload/3575/3575fig2.jpg" alt="図2" /> 図2。シミュレートされた虚血後の異なる細胞集団の代表は、ドットプロット。 いくつかの実験の評価翔虚血後のH9c2のcardiomyoblastに間葉系幹細胞の添加は虚血後に単独で培養した心筋芽細胞（図3）と比較して有意に生きている細胞の割合を増加させることを結婚する。 <img src="/files/ftp_upload/3575/3575fig3.jpg" alt="図3" /> 図3。虚血後のcardiomyoblasts上に幹細胞治療の効果は生きている細胞の割合が大幅にシミュレートされた虚血後に減少した対照と比較して（制御対IRモデル：90.36 ± 2.60対10.52 ± 0.48、*** P&gt; 0.001）。、添加後の増加間葉系幹細胞の（IRモデル対IR + BMSC：10.52 ± 0.48対25.21 ± 2.13、*** P&gt; 0.001）。データは平均値± SEMを表す。データの統計解析は、Tukeyの多重比較の事後検定と一元配置分散分析を用いて行った。

<ol>
	<li>Dimmeler, S., Burchfield, J., Zeiher, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Therapy+of+Myocardial+Infarction.">Cell-Based Therapy of Myocardial Infarction.</a> Arterioscler Thromb Vasc Biol. 28, (2008).</li><li>Kim, S. U., de Vellis, J. <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+cell+therapy+in+neurological+diseases:+A+review.">Stem cell-based cell therapy in neurological diseases: A review.</a> Journal of Neuroscience Research. 87, 2183-21 (2009).</li><li>Lee, K., Chan, C. K., Patil, N., Goodman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+for+bone+regeneration-Bench+to+bedside.">Cell therapy for bone regeneration-Bench to bedside.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. 89, 252-25 (2009).</li><li>Gaipa, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMP-based+CD133++cells+isolation+maintains+progenitor+angiogenic+properties+and+enhances+standardization+in+cardiovascular+cell+therapy.">GMP-based CD133+ cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy.</a> Journal of Cellular and Molecular Medicine. 14, 1619-1619 (2010).</li><li>Trounson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+in+human+stem+cell+therapeutic+research.">New perspectives in human stem cell therapeutic research.</a> BMC medicine. 7, 29-29 (2009).</li><li>Mazhari, R., Hare, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+action+of+mesenchymal+stem+cells+in+cardiac+repair:+potential+influences+on+the+cardiac+stem+cell+niche.">Mechanisms of action of mesenchymal stem cells in cardiac repair: potential influences on the cardiac stem cell niche.</a> Nat Clin Pract Cardiovasc Med. 4, S21-S21 (2007).</li><li>Kimes, B. W., Brandt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+a+clonal+muscle+cell+line+from+rat+heart.">Properties of a clonal muscle cell line from rat heart.</a> Experimental Cell Research. 98, 367-367 (1976).</li><li>Sardao, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+imaging+of+H9c2+myoblasts+exposed+to+tert-butylhydroperoxide--characterization+of+morphological+features+of+cell+death.">Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide--characterization of morphological features of cell death.</a> BMC Cell Biol. 8, 11-11 (2007).</li><li>Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological,+biochemical,+and+electrophysiological+characterization+of+a+clonal+cell+(H9c2)+line+from+rat+heart.">Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.</a> Circulation research. 69, 1476-1476 (1991).</li><li>Cselenyak, A. <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+rescue+cardiomyoblasts+from+cell+death+in+an+in+vitro+ischemia+model+via+direct+cell-to-cell+connections.">Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections.</a> BMC Cell Biol. 11, 29-29 (2010).</li><li>Sauvant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+an+in+vitro+model+system+for+investigation+of+reperfusion+damage+after+renal+ischemia.">Implementation of an in vitro model system for investigation of reperfusion damage after renal ischemia.</a> Cell Physiol Biochem. 24, 567-567 (2009).</li><li>Namas, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-Induced+Overexpression+of+BNIP3+is+Not+Dependent+on+Hypoxia-Inducible+Factor+1alpha+in+Mouse+Hepatocytes.">Hypoxia-Induced Overexpression of BNIP3 is Not Dependent on Hypoxia-Inducible Factor 1alpha in Mouse Hepatocytes.</a> Shock. 36, 196-196 (2011).</li><li>Cao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia/Reoxygenation+Up-Regulated+the+Expression+of+Death+Receptor+5+and+Enhanced+Apoptosis+in+Human+Hepatocyte+Line.">Hypoxia/Reoxygenation Up-Regulated the Expression of Death Receptor 5 and Enhanced Apoptosis in Human Hepatocyte Line.</a> Transplantation Proceedings. 38, 2207-2207 (2006).</li><li>Meloni, B. P., Meade, A. J., Kitikomolsuk, D., Knuckey, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+neuronal+cell+death+in+acute+and+delayed+in+vitro+ischemia+(oxygen-glucose+deprivation)+models.">Characterisation of neuronal cell death in acute and delayed in vitro ischemia (oxygen-glucose deprivation) models.</a> Journal of Neuroscience Methods. 195, 67-67 (2011).</li><li>Mimeault, M., Hauke, R., Batra, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+a+revolution+in+therapeutics--recent+advances+in+stem+cell+biology+and+their+therapeutic+applications+in+regenerative+medicine+and+cancer+therapies.">Stem cells: a revolution in therapeutics--recent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies.</a> Clinical Pharmacology &#38; Therapeutics. 82, 252-252 (2007).</li><li>Angoulvant, D. <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+cell+conditioned+media+attenuates+in+vitro+and+ex+vivo+myocardial+reperfusion+injury.">Mesenchymal stem cell conditioned media attenuates in vitro and ex vivo myocardial reperfusion injury.</a> J Heart Lung Transplant. 30, 95-95 (2010).</li><li>Lim, Y. J., Zheng, S., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+Preconditions+Purkinje+Cells+against+Cell+Death+under+In+Vitro+Simulated+Ischemia/Reperfusion+Conditions.">Morphine Preconditions Purkinje Cells against Cell Death under In Vitro Simulated Ischemia/Reperfusion Conditions.</a> Anesthesiology. 100, 562-562 (2004).</li><li>Guo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-receptor-mediated+protection+of+cerebral+endothelial+cell+viability+and+mitochondrial+function+after+ischemic+insult+in+vitro.">Estrogen-receptor-mediated protection of cerebral endothelial cell viability and mitochondrial function after ischemic insult in vitro.</a> J Cereb Blood Flow Metab. 30, 545-545 (2010).</li></ol>

利害の衝突は宣言されません。

実証プロトコルはそのようなモデルのすべての利点と欠点と心筋梗塞の幹細胞治療、のはるかに複雑な問題へのin vitroでのアプローチになります。もちろん、それが心筋梗塞の間と後に起きている複雑な（例えば、免疫学）のイベントを反映することはできませんが、虚血後の細胞で追加された細胞の直接的な効果に焦点を当てることができます。 H9c2 cardiomyoblasts上でシミュレートされた虚?...

<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 </td></tr><tr><td>カルセイン- AM </td><td>分子プローブ</td><td> L3224、C3099 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td>エチジウムホモ二量体A - 2 </td><td>分子プローブ</td><td> L3224、E3599 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> Vybrantは、DID </td><td>分子プローブ</td><td> V22887 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr></tbody></table> 表1。試薬。 <table border="1"><tbody><tr><td> 機器の名前 </td><td> 会社 </td><td> コメント（省略可能） </td></tr><tr><td>ツァイス顕微鏡用PeCon細胞培養システム</td><td> PeCon社</td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> 表2。機器。

stem cell transplantation in an in vitro simulated ischemia/reperfusion model

幹細胞移植のプロトコルは、臨床実践1,2,3への道を模索しています。 、より良い結果を得るためのプロトコルは、より堅牢な作り、そして埋め込 ​​み型細胞の新しい源を見つけることは4,5最近の研究の焦点です。細胞治療の有効性を調査することは容易ではないとの新しいツールは、処理工程6に関与するメカニズムを調査するために必要とされる。我々は、虚血/再灌流傷害時に、幹細胞移植後の携帯電話接続、さらには細胞内メカニズムの観察を可能にすると細胞療法の有効性を評価するために、虚血/再灌流の実験的なプロトコルを設計した。 H9c2 cardiomyoblast細胞は、細胞培養プレート7,8の上に置かれた。虚血は0.5％以下の酸素レベルとグルコースを含まない培地で150分でシミュレートされました。その後、通常のメディアと酸素レベルは、再灌流をシミュレートするために再導入されました。酸素グルコース欠乏後に、損傷した細胞を培養するためにそれらを追加することにより、間葉系幹細胞由来の標識されたヒトの骨髄の移植で治療された。彼らは簡単に拡張および自家、最小侵襲手術で簡単にアクセス可能であるため、間葉系幹細胞が臨床試験で好ましい。共培養の24時間後、細胞は生細胞と死細胞を区別するためカルセインとエチ-ホモ二量体で染色した。このセットアップでは、私たちは共焦点蛍光顕微鏡を用いて細胞間の接続を調査し、フローサイトメトリーによる虚血後の細胞の生存率を定量化することができました。共焦点顕微鏡は、細胞融合と細胞間のナノチューブの形成などの2つの細胞集団の相互作用を示した。フローサイトメトリー分析は、グラフ上にプロットし、統計的に分析することができる損傷した細胞の3つのクラスタを明らかにした。これらの集団が別々に調べることができると結論は、シミュレートの有効性にこれらのデータを描画することができます治療的アプローチ。

Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

我々は、セットアップする方法を示しています<em&gt; in vitroで</em&gt;虚血/再灌流モデルとどのように虚血後の心筋細胞の幹細胞治療の効果を評価する。

における幹細胞移植 in vitroで模擬虚血/再灌流モデル

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

stem-cell-transplantation-an-vitro-simulated-ischemiareperfusion

Research

JoVE Journal

Medicine

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

14.0K ビュー.  Semmelweis University. 我々は、セットアップする方法を示しています in vitroで虚血/再灌流モデルとどのように虚血後の心筋細胞の幹細胞治療の効果を評価する。

ビデオ: Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

のマウスモデル子宮内で移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

医学

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

マウスにおける急性腎障害とポスト傷害線維の虚血再灌流モデル

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

脊髄損傷の実験的挫傷モデルにおける神経幹細胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

などの酸素グルコース欠乏および再酸素化<em&gt;インビトロ</em&gt;虚血再灌流傷害モデル血液脳関門の機能障害を研究するための

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

腸管虚血 - 再灌流傷害のマウスモデル

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

作成および糖尿病創傷治癒モデルにおける脂肪由来幹細胞 (ASC) シートの移植

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

2 血管閉塞マウス脳虚血・再灌流モデル

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

再建微小外科における虚血再灌流損傷の研究のための前臨床ラットモデル

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...