著者はfluc/mrfp/sr39tkレポーター遺伝子の贈り物をSanjivガンビールに感謝したいと思います。この作品は、カナダの幹細胞ネットワーク（SCN）とジェシーの旅財団によって資金を供給された。

1。 C2C12筋芽細胞の維持と伝播<ol><li>プレート75cm 2フラスコでC2C12筋芽細胞および10％の最終濃度になるようにウシ胎児血清（FBS）を添加した高グルコースダルベッコ改変イーグル血清（HG-DMEM）中で細胞を維持。これは筋芽細胞の人口を激減させるように細胞がいつでもコンフルエントになることを許可しないようにしてください。媒体は、隔日に変更する必要があります注：常に暖かい培地を37℃にして、使用する前に水浴のC。 </li><li>筋芽細胞が約80％コンフルエントになったときに、新しいフラスコに継代細胞。培地を吸引します。トリプシン阻害血清を含む培地のすべてのトレースを削除する4〜5ミリリットルハンクス平衡塩溶液（HBSS）で細胞を洗浄します。簡単に言えば2〜3ミリリットル0.25％（w / v）のフラスコから付着筋芽細胞を解離するトリプシン-EDTA溶液で細胞層を洗浄してください。 5分間37℃でインキュベーター内でトリプシンと場所フラスコを吸引します。 </li><li>このインキュベーション中にるには、HG-DMEM/10％FBS培地9mlの持つ新しいフラスコを準備します。トリプシン処理後、培地内のすべてのセルのコレクションを確実にするために細胞やピペット4-5回に完全培地10 mlを加える。新しいフラスコに細胞懸濁液1 mlを加え、37℃、5％CO 2中でインキュベート℃、 </li></ol> 2。 C2C12細胞トランスフェクション<ol><li>一度細胞がコンフルエントに50％に達しており、Invitrogenからメーカーの指示に従ってトランスフェクション。簡単に言えば、4.5ミリリットルのOPTI-MEM培地で90μlのリポフェクタミン2000試薬を組み合わせています。別のチューブに、4.5ミリリットルのOPTI-MEMで36μgのCMV-trifusionレポーター遺伝子DNAを兼ね備えています。各チューブは5分を組み合わせて、待つことにフリックします。穏やかに混合し、正確に30分間室温でインキュベートし、両方のチューブの内容を結合注：第二のフラスコものみリポフェクタミンおよびOPTI-MEMはネガティブコントロールとして機能するように、受信トランスフェクトしていないセルに対して設定する必要があります。 </li><li> C2C12細胞から培地を除去し、15.5を追加新鮮HG-DMEM/10％FBS培地1ml。 20mlの総容積を持って来るために、トランスフェクション培地を追加します。 </li><li>細胞は37℃で少なくとも20時間一晩トランスフェクトすることができ℃、 </li><li>翌日、トランスフェクション培地を吸引し、10ミリリットルHG-DMEM/10％FBSを追加します。 </li><li>倒立蛍光顕微鏡下で細胞を見る。明視野および赤色蛍光画像（; 607分の584 nmのMRFP元/全角TRITCフィルターキューブを使用）の両方をキャプチャします。トランスフェクション効率を生成するために、ビューの複数のフィールドの明るいフィールドの下で見た細胞の総数で割った蛍光下で観察し、RFPを発現している細胞の数をカウントします。 </li></ol> 3。細胞生存/ MTTアッセイの評価<ol><li> 24ウェルプレートでのトランスフェクションは、プレートの各ウェルに1×10 5 C2C12細胞の前に一日。細胞はHG-DMEM/10％FBS中500μlの体積に播種する必要があります。 </li><li>翌日、前述のように一晩トランスフェ芽。トランスフェクションreagenについては、メーカーの提案に従う24ウェルプレート用のトンボリューム。コントロールとしてのみリポフェクタミン（DNAなし）で井戸のセットをインキュベートする。その適切なトランスフェクションが行われたことを確認するために蛍光で細胞を見る。 </li><li> HG-DMEM/10％FBS中5 mg / mlのチアゾリルブルーテトラゾリウムブロミド（MTT）を用いてトランスフェクション培地でインキュベート筋芽細胞を取り除きます。トランスフェクトされた井戸の8にD-ルシフェリンを追加し、4時間37℃でインキュベートする。 </li><li>井戸からメディアを取り出します。各ウェルに180μlのイソプロパノールを添加することにより、青色ホルマザン結晶を可溶化する。 15分間37℃で振とうする。 </li><li> 96ウェルプレートに沈殿物、ピペット溶液の回避および575 nmの吸光度を読み取る。 </li></ol> 4。移植のための筋芽細胞の調製<ol><li> 、培地を除去HBSSで筋芽細胞を洗浄し、1.1で説明したように、細胞をトリプシン処理。 </li><li>完全培地4mlに細胞を再懸濁する。 </li><li>血球計を使用して、10 6芽細胞を含むボリュームを生成する細胞を数える。ピペットVOL滅菌済みの1.5mlマイクロチューブに梅。 〜10％のトランスフェクション効率と、これらの値は、100,000ルシフェラーゼ発現細胞は、移植後のGE ExploreOptixスキャナで検出可能であることを示している。 </li><li> 10 6 C2C12細胞を含む、15μlの最終噴射量を達成する。必要であれば、1分間2,000 rpmでマイクロチューブを遠心します。ピペットで注意深く上清を吸引します。 HG-DMEMにFBSを欠く15μlの中に細胞を再懸濁する。 </li></ol> 5。細胞移植<ol><li>イソフルラン2パーセント/ 2％O 2でマウスを麻酔。背側後肢エリアから毛をむしる。イソフルラン1.5パーセント/ 2％のO 2で麻酔を維持します。 </li><li> C2C12筋芽細胞をよく懸濁していることを確認します。腹臥位でマウスを使って、後肢を拡張し、30°の角度で腓腹筋の外側頭に直接細胞を注入するためにインスリン注射器を使用しています。右後肢とトランスフェクトしていない筋芽細胞のintにトランスフェクトされた筋芽細胞を注入対側（左）後肢をO。 </li><li>ベースライン生物発光スキャンを実行：すばやく腹臥位でマウスを敷設、光スキャナのステージにマウスを移す。麻酔ラインをフックアップ。マウスを確保するために麻酔をかけたまま、二人目は、スキャナの他の場所の動物の間に麻酔ラインをフックするために存在している必要があります。 </li><li>注射の面積が表示されるように優しく後肢を拡張します。そのような医療用テープなど優しい接着剤のある場所でテープ後肢。 </li><li>これが検出されたバックグラウンド信号を増加させるように、チャンバーを閉じて、光がスキャナ内部にアクセスすることはできませんことを確認してください。スキャナは、生物発光イメージングについては、製造元の指示に含まれるパラメータに設定する必要があります。具体的には、 &quot;全くレーザー&quot;が選択されていないことを確認してください。 </li><li>羽をむしられた領域の周囲に関心領域（ROI）を描き、スキャンを開始します。 </li></ol> 6。 Fluc基質の注入、mdxマウスにD-ルシフェリン、 &lt;OL&gt; <li>マウスに麻酔を腹腔内にある間にホタルルシフェラーゼ基質D-ルシフェリン（40 mg / mlの原液から、製造元の指示に従って構成される）の150 mg / kgを注入します。 </li><li>マウスを回復して、次のスキャンのためにマウスを準備する前に15分の取り込み期間を可能にします。 </li></ol> 7。 DMDのマウスモデルにインプラントを次のターゲットリュック発現のMPCへのBLI <ol><li> 5.1で説明されるように摂取期間の後、再びマウスを麻酔。 </li><li>光学スキャナに戻ってマウスを転送し、バックグラウンドでのスキャンと同様に別の生物発光スキャンを実行します。 </li><li> 20分取込期間が最大信号強度を提供する必要がありますが、その後のスキャンを実行することができる。 </li><li>画像収集が完了すると、施設内動物倫理委員会及びアニマル·ケアに関するカナダ評議会（CCAC）によって設定されたガイドラインに沿ってマウスを生け贄に捧げる。私はすぐに後肢の筋肉や場所を隔離パラフィン包埋のために修正するためにnを10％ホルマリン。筋芽細胞の筋肉内注射を確認するルシフェラーゼの免疫組織化学染色を行います。 </li></ol>

<ol>
	<li>Emery, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+muscular+dystrophies.">The muscular dystrophies.</a> Lancet. 359, 687-695 (2002).</li><li>Blake, D. 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=Function+and+genetics+of+dystrophin+and+dystrophin-related+proteins+in+muscle.">Function and genetics of dystrophin and dystrophin-related proteins in muscle.</a> Physiol. Rev. 82, 291-329 (2002).</li><li>Goldring, K., Partridge, T., Watt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> J. Pathol. 197, 457-467 (2002).</li><li>Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+that+participate+in+regeneration+of+skeletal+muscle.">Cells that participate in regeneration of skeletal muscle.</a> Gene Ther. 9, 752-753 (2002).</li><li>Moss, F. P., Leblond, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+as+the+source+of+nuclei+in+muscles+of+growing+rats.">Satellite cells as the source of nuclei in muscles of growing rats.</a> Anat. Rec. 170, 421-435 (1971).</li><li>Snow, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autoradiographic+study+of+satellite+cell+differentiation+into+regenerating+myotubes+following+transplantation+of+muscles+in+young+rats.">An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats.</a> Cell Tissue Res. 186, 535-540 (1978).</li><li>Kuang, S., Rudnicki, M. 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+emerging+biology+of+satellite+cells+and+their+therapeutic+potential.">The emerging biology of satellite cells and their therapeutic potential.</a> Trends Mol. Med. 14 (2), 82 (2008).</li><li>Hoffman, E. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+lights.">Molecular imaging in living subjects: seeing fundamental biological processes in a new lights.</a> Genes Dev. 17, 545-580 (2003).</li><li>Sicinski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+muscular+dystrophy+in+the+mdx+mouse:+a+point+mutation.">The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.</a> Science. 244, 1578-1580 (1989).</li><li>Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathogenesis of neurodegenerative disorders. In: Contemporary neuroscience. X, 294 (2001).</li><li>Andersson, J. E., Bressler, B. H., Ovalle, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+regeneration+in+hind+limb+skeletal+muscle+of+the+mdx+mouse.">Functional regeneration in hind limb skeletal muscle of the mdx mouse.</a> J. Muscle Res. Cell Motil. 9, 499-515 (1988).</li></ol>

著者らは、開示することは何もない。

そこで本研究では、移植後の非侵襲的に標的筋芽細胞/ MPCのための高速かつ信頼性の高い分子イメージング、レポーター遺伝子アプローチを説明してきました。この研究はbioluminescenseイメージング（BLI）を介して移植されたMPCの短期的な局在を示しているが、細胞が標的とされる方法は 、実際には、簡単に安定して発現していることが細胞の移植による細胞移植の長期的評価に?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> C2C12筋芽細胞</td><td> ATCC </td><td></td><td></td></tr><tr><td>ダルベッコ改変イーグル培地</td><td>ライフテクノロジーズ</td><td> 12800-017 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>ライフテクノロジーズ</td><td> 12483020 </td><td></td></tr><tr><td> 0.25％トリプシン-EDTA </td><td>ライフテクノロジーズ</td><td> 25200から72 </td><td></td></tr><tr><td>ハンクス平衡塩溶液</td><td>シグマアルドリッチ</td><td> H6648 </td><td></td></tr><tr><td>リポフェクタミン2000 </td><td>ライフテクノロジーズ</td><td> 11668-019 </td><td></td></tr><tr><td>ニコンエクリプスTE2000-5 </td><td>ニコンインスツル株式会社</td><td></td><td></td></tr><tr><td> Xenolight D-ルシフェリン</td><td>パーキンエルマー</td><td> 122796 </td><td> PBS中40 mg / mlの</td></tr></tbody></table>

molecular imaging to target transplanted muscle progenitor cells

デュシェンヌ型筋ジストロフィー（DMD）は3500男子 ​​で1に影響を及ぼす重篤な遺伝性神経筋疾患であり、進行性筋変性1,2によって特徴づけられる。患者では、損傷した筋線維を再生させる常駐筋衛星細胞の能力（SCS）は4ますます効率が悪くなる。したがって、健常者の筋前駆細胞（MPCは）/筋芽細胞の移植は、DMDに対する有望な治療アプローチである。幹細胞療法の使用への主要な制限は、しかし、移植細胞の長期的なモニタリングのため、その有効性を評価するための信頼性の高い画像処理技術の欠如である。ここでは、筋芽細胞移植の成功を評価するための非侵襲、リアルタイムのアプローチについて説明します。この方法は、（ホタルルシフェラーゼ[fluc]、単量体赤色蛍光タンパク質[MRFP]とSR39チミジンキナーゼ[sr39tk]）expre遺伝子から成る統一融合レポーター遺伝子を利用していますssionは異なる撮像モダリティ9、10に結像させることができる。陽電子放射断層撮影（PET）、単光子放出コンピュータ断層撮影（SPECT）、磁気共鳴イメージング（MRI）、光学イメージング、および高周波3次元超音波を含むイメージングモダリティ、様々なユニークな長所、短所がそれぞれ、利用可能になりました11。生物発光イメージング（BLI）の研究では、例えば、比較的低コスト、高スループットであるという利点を持っています。これは、本研究では、我々はマウスに移植後の生存能力のあるC2C12筋芽細胞の短期のローカリゼーションのための融合遺伝子と生物発光イメージング（BLI）内に含まれるホタルルシフェラーゼ（fluc）レポーター遺伝子配列を利用して、このような理由からですDMDのモデル12-14（X染色体[MDX]マウスの筋ジストロフィー）。重要なのは、BLIは、標識されたMPCは、移植後の体内動態を検討するための手段を提供してくれますし、tで繰り返し細胞を追跡するために有用であろうIMEとは、次の移行。私たちのレポーター遺伝子アプローチはさらに、私たちは、単一の生体内で複数の画像診断法をマージすることができ、断層の性質、空間分解能と大きな動物とヒト10,11にスケールアップする能力を考えると、PETは、私たちが将来の仕事の基礎を形成します前臨床モデルへと臨床応用への細胞内で開発された方法の迅速な翻訳を促進することが示唆された。

50から60パーセントコンフルエント時に、C2C12筋芽細胞を一過上記融合レポーター遺伝子はホタルルシフェラーゼ成る構築物でトランスフェクトした[fluc]、単量体の赤色蛍光タンパク質[MRFP]とSR39チミジンキナーゼ[sr39tk]（ 図1A）。トランスフェクション効率を構築する私たちのレポーターにMRFPシーケンスを利用して、蛍光顕微鏡（ 図1B、C）...

Watch this Scientific Journal Video about Molecular Imaging to Target Transplanted Muscle Progenitor Cells at JoVE.com

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

筋芽細胞移植の成功を評価するための非侵襲的な手段が記載されている。この方法は、その発現が異なる撮像モダリティで撮影することができる遺伝子を統合的融合レポーター遺伝子を利用しています。ここでは、利用すること<em&gt; fluc</em生物発光イメージングを介して細胞を標的とする&gt;レポーター遺伝子配列。

移植筋前駆細胞を標的とする分子イメージング

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

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9.1K ビュー.  Lawson Health Research Institute. 筋芽細胞移植の成功を評価するための非侵襲的な手段が記載されている。この方法は、その発現が異なる撮像モダリティで撮影することができる遺伝子を統合的融合レポーター遺伝子を利用しています。ここでは、利用すること flucレポーター遺伝子配列。

ビデオ: Molecular Imaging to Target Transplanted Muscle Progenitor Cells

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

炎症性プラークの赤外蛍光（NIRF）血管内分子イメージング、マルチモーダルアプローチの近くin vivoでの動脈硬化のイメージングに

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

医学

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

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

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 ...

The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically inactive for prolonged periods of time, and begin with an unexpected bout of exercise1-4, but can also occur in athletes who exercise beyond their normal limits of training5. The symptoms associated with this painful phenomenon can range from slight muscle tenderness, to severe debilitating pain1,3,5. The intensity of these symptoms and the related discomfort increases within the first 24 hours following the termination of the exercise, and peaks between 24 to 72 hours post exercise1,3. For this reason, DOMS is one of the most common recurrent forms of sports injury that can affect an individual&#x2019;s performance, and become intimidating for many1,4.
For the last 3 decades, the DOMS phenomenon has gained a considerable amount of interest amongst researchers and specialists in exercise physiology, sports, and rehabilitation fields6. There has been a variety of published studies investigating this painful occurrence in regards to its underlying mechanisms, treatment interventions, and preventive strategies1-5,7-12. However, it is evident from the literature that DOMS is not an easy pathology to quantify, as there is a wide amount of variability between the measurement tools and methods used to quantify this condition6. It is obvious that no agreement has been made on one best evaluation measure for DOMS, which makes it difficult to verify whether a specific intervention really helps in decreasing the symptoms associated with this type of soreness or not. Thus, DOMS can be seen as somewhat ambiguous, because many studies depend on measuring soreness using a visual analog scale (VAS)10,13-15, which is a subjective rather than an objective measure. Even though needle biopsies of the muscle, and blood levels of myofibre proteins might be considered a gold standard to some6, large variations in some of these blood proteins have been documented 6,16, in addition to the high risks sometimes associated with invasive techniques. 
Therefore, in the current investigation, we tested a thermal infra-red (IR) imaging technique of the skin above the exercised muscle to detect the associated muscle soreness. Infra-red thermography has been used, and found to be successful in detecting different types of diseases and infections since the 1950&#x2019;s17. But surprisingly, near to nothing has been done on DOMS and changes in skin temperature. The main purpose of this investigation was to examine changes in DOMS using this safe and non-invasive technique.

The purpose of this investigation was to assess whether using an infra-red thermal camera is a valid tool for detecting and quantifying the muscle soreness after exercising.

遅発性筋痛を検出する熱赤外線イメージングの使用

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

低分子CLT1ペプチド標的造影剤を用いて前立腺がんの分子イメージングのMR

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

ハイスループットフローサイトメトリー細胞ベースのアッセイN-メチル-D-アスパラギン酸受容体またはヒト血清中のドーパ​​ミン-2受容体に対する抗体を検出する

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

シーケンシャル<em&gt;インビボ</em&gt;骨折修復中に、骨形成幹細胞/前駆細胞のイメージング

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨格筋疾患の定量的磁気共鳴イメージング

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

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 ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

血管平滑筋細胞と大動脈石灰化と炎症のイメージングの石灰化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

援助医薬品開発への人間の子宮平滑筋の収縮力測定

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...