ティムドイル博士と生物発光イメージングの支援のためのin vivoイメージングのためのスタンフォードセンターに感謝します。また、マトリックス溶液と幹細胞の共同注射で彼女のテクニックを共有するためのパンガン黄、博士のおかげ。最後に、スティーブのおかげで、博士号を取得してフェルト獣医、動物のケアと支援のための。

<ol><li> ダブル融合レポーター遺伝子の構築 <ol><li>ヒト胚性幹細胞の生物発光イメージングを行うためには、まず安定的にそのようなユビキチンまたはEF1aのような構成的プロモーターによって駆動されるホタルルシフェラーゼなどのルシフェラーゼレポーター遺伝子を発現する細胞を取得する必要があります。 </li><li>このプロトコルの焦点は、レポーター遺伝子のアプリケーション上にある、詳細な手順はここで提供されていないので。しかし、私たちの研究室の一般的な戦略は、pCDNA 3.1内スペーサー+で区切られたホタルルシフェラーゼ（変動を確認）と強化された緑色蛍光タンパク質（EGFP）を含むベクターを構築ダブルフュージョンを使用することです。 </li><li>簡単に言えば、私たちの二重融合遺伝子は、もともとpCDNA 3.1のサイトメガロウイルスプロモーター（+）の下流に位置していたので、3.3 kbpの断片を自己不活性化（SINのマルチクローニングサイトにライゲーションをNdeIおよびNotIで消化し、平滑末端を用いて切り出し、ユビキチンプロモーターの制御下にある）レンチウイルスベクター、。 </li></ol></li><li> レンチウイルス形質導入 <ol><li>ヒトES細胞は、10の感染多重度（MOI）（106細胞とインキュベートした約107個のウイルス力価）で継代した後3-5日を形質導入することができます。 </li><li>融解ウイルスストックは、新鮮なhESCのメディアに直接追加することができます。 </li><li>媒体12および24時間後をリフレッシュします。 </li><li> 48時間後、形質導入効率はによって定性的に蛍光顕微鏡を用いて評価することができます。その後、FACSは、感染細胞を単離するために使用することができます。 </li></ol></li><li> 培養ヒトESおよびセットアップイメージング入門 <ol><li>の文化あなたのルシフェラーゼ正ヒトES細胞をフィーダーフリーの条件。我々は通常、標準WiCellのプロトコルに従い、馴化培地または商用のいずれかを使用してマトリゲル削減成長因子でコーティングした6ウェルプレート、、フィーダーフリーメディアを使用してください。 </li><li>ルシフェラーゼ陽性細胞を検出するには、レポータープローブD -ルシフェリンがすでに準備されている必要があります。 45 mg / mLの最終濃度が1から1.5 mLの調製物中の上澄みルシフェリン、これを行うには。時ではないストレージにペーパータオルで覆うことにより、光への曝露を使用して回避しないで時-20℃で分注してください。 </li><li>ルシフェラーゼ陽性細胞を可視化するために、我々は、IVIS 50、IVIS 200イメージングシステムを（Xenogen社、アラメダ、カリフォルニア州）を使用します。この後者のシステムでは、小動物の一時的な麻酔のための統合isoflourane装置と誘導室が含まれています。 </li></ol></li><li> ヒトES細胞のin vitroでの生物発光イメージングに <ol><li> in vitroでの生物発光イメージングのためには、無菌状態を維持することが重要です。したがって、イメージングシステムは、好ましくは細胞培養室で、無菌でなければなりません。 </li><li>イメージングの前に、細胞培地を除去し、細胞をカバーするだけの十分なPBSを加える。たとえば、hESCの文化を含む6ウェルプレートの各ウェルに1mLのPBSを加える。 </li><li> PBSにD -ルシフェリンの比率は、6ウェルプレートの各ウェルに解凍したD -ルシフェリンの10μLを加えるので、1:100である必要があります。 </li><li> 1分間待機し、その後1秒の露光時間を使用してイメージを取る。信号が弱い場合は、露出の間隔を長くして再試行してください。 </li><li>生物発光シグナルは、細胞数を反映し、定量アッセイが異なる細胞数で信号を相互に関連付けることが実行できるように。 </li></ol></li><li> in vivoイメージングのためのマウスと注入細胞の調製 <ol><li>レポーター遺伝子イメージングは​​日、週、そしてさらに数ヶ月にわたって移植後の細胞を追跡するため、ハイスループット、安価で感度の高い方法を提供する。移植された幹細胞は、頻繁に、奇形腫を形成する宿主の免疫システムによって拒絶されている、または単に死ぬので、これは特に重要です。 </li><li> in vivoでのイメージング奇形腫形成のための最も簡単な方法は、SCIDマウスの背部にホタルルシフェラーゼレポーター遺伝子を発現し、IVISシステムで生物発光画像を取得するヒトES細胞を注入することです。 </li><li>あなたが細胞を移植する準備ができたら、、細胞を緩めて数回洗浄し、そしてDMEM -マトリゲルを削減成長因子の1:1混合物でそれらを中断するディスパーゼまたはコラゲナーゼを使用してください。一般的に我々は最初のチルドDMEMで細胞を混合し、マトリゲルに追加してください。それぞれの皮下注射部位では、マトリゲル/ DMEM混合物の50〜100μLの容積に懸濁した20万セルを注入する。細胞数は、アプリケーションに応じて調整することができます。注射前に氷の上のすべてを維持してください。 </li><li>細胞が準備ができたら、オンにイソフルランフローとインダクションボックスに置くことによって、マウスを麻酔。 1〜5分後、マウスが眠っていると、連続イソフルランと手術台上に配置することができます。 </li><li>毛皮自然に自動蛍光を発するとは、生物発光画像を覆い隠す可能性があるため、我々は、マウスの裏面から毛を削除する必要があります。これを行う最も簡単な方法は、電気シェーバーを使用することですが、あなたも脱毛のゲルを使用することができます。滅菌にアルコールで拭いてくださいその後皮膚</li><li>次に、注射器で細胞懸濁液を描く。彼らは細胞を詰まらせることはありませんので、23から27ゲージの針が最適です。針は、（&lt;23ゲージ）が大きすぎる場合、針管は、細胞が戻って漏れるできるような大きな穴が残る場合があります。 </li><li>マウスの背中に皮膚の下に細胞を注入する。肌がかなり緩んでいるので、挟まないように親指と人差し指を使用し、注入する領域を差し伸べる。あまりにも深く穿刺するように注意していないながら、皮膚のすぐ下に注入する。また、プランジャーを押しながら注射部位から滑り落ちるから針を維持しようとする：これは再び挿入しようとすると、細胞がリークすることができますを介して皮膚に穴を作成できなくなります。 </li><li>細胞が注入された後、マウスが目を覚ますと生物発光イメージングのために再麻酔の前に周りの実行を許可するように数時間を待つ。そうすることで、イソフルラン毒性を回避することができます。 </li></ol></li><li> 移植細胞のin vivo生物発光イメージングに <ol><li>全体の動物の生物発光イメージングのために、我々は、375 mg / kg体重でのD -ルシフェリンを腹腔内注射を行います。注射前に投与量を計算するために動物を量る。 </li><li>ちょうど正中線から、腹膜にD -ルシフェリンを注入する。あまり深く行かないしたり、内臓に損傷を与えることができるように注意してください。マウスが目を覚ますに始まる場合は、ノックアウトのボックスに戻ってそれを配置し、1〜2分待つ。マウスの場合、最大の生物発光は、通常、注射後15〜40分を発生します。 </li><li>ヒトES細胞から放出されるではない任意の光を吸収するためのイメージングボックスにマット黒い紙を置くことを忘れないでください。黒い紙の上に、イソフルランの撮像室内でマウス（裏面を上にして）置きます。 10秒の露光時間で始まります。信号が飽和している場合、露光時間を減らしてみてください。弱すぎる場合、露出を増加させる。 </li><li>信号の露光時間が、あなたのマウスに最適化されると、信号が最大値に達するまで、毎分の画像を撮影開始。これは最適な注射部位をカバーする&quot;関心領域（ROI）&quot;を選択するイメージングソフトウェアを使用して行われます。各ROIでの信号強度を監視することにより、簡単に最大の信号強度に達したことを示す、信号が低下し始める時に決定することができます。最大信号は、最終的なデータとして使用する必要があります。 </li><li>あなたがあなたのイメージに問題がなければ、イソフルランからマウスを削除し、そのケージに目を覚ますことができます。通常、マウスは15分以内に目覚めるはず。 </li><li>ヒトES細胞は動物の中で生き残るように生物発光レポーター遺伝子イメージングは​​、長い間、毎日、毎週、毎月繰り返すことができます。ヒトES細胞が増殖し始めると、発展途上奇形腫からの信号は、通常、数週間のために、時間の経過とともに指数関数的に増加します。 </li><li>生物発光画像の所望の時間経過を取得した後、動物を屠殺し、組織切片は、組織学のために使用することができます。 </li></ol></li></ol>

<ol>
	<li>Passier, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cardiomyocyte+differentiation+from+human+embryonic+stem+cells+in+serum-free+cultures.">Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.</a> Stem Cells. 23, 772-780 (2003).</li><li>Laflamme, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocytes+derived+from+human+embryonic+stem+cells+in+pro-survival+factors+enhance+function+of+infarcted+rat+hearts.">Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.</a> Nature biotechnology. 25, 1015-1024 (2007).</li><li>Baharvand, H., 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=Neural+differentiation+from+human+embryonic+stem+cells+in+a+defined+adherent+culture+conditi.">Neural differentiation from human embryonic stem cells in a defined adherent culture conditi.</a> Int J Dev Biol. 51, 371-378 (2007).</li><li>Schulz, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neuronal+differentiation+of+human+embryonic+stem+cells.">Directed neuronal differentiation of human embryonic stem cells.</a> BMC Neurosci. 4, (2007).</li><li>Jiang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Insulin-producing+Islet-like+Clusters+from+Human+Embryonic+Stem+Cells.">Generation of Insulin-producing Islet-like Clusters from Human Embryonic Stem Cells.</a> Stem Cells. 17, 17 (2007).</li><li>Swijnenburg, R. J., van der Bogt, K. E. A., Sheikh, A. Y., Cao, F., 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=Clinical+hurdles+for+the+transplantation+of+cardiomyocytes+derived+from+human+embryonic+stem+cells:+role+of+molecular+imaging.">Clinical hurdles for the transplantation of cardiomyocytes derived from human embryonic stem cells: role of molecular imaging.</a> Current Opinion in Biotechnology. 18, 38-45 (2007).</li><li>Herschman, H. R. <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:+looking+at+problems,+seeing+solutions.">Molecular imaging: looking at problems, seeing solutions.</a> Science. 302, 605-608 (2003).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+fluorescent+protein+markers+in+living+cells.">Development and use of fluorescent protein markers in living cells.</a> Science. 300, 87-91 (2003).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature medicine. 4, 245-247 (1998).</li><li>Bhaumik, S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+Renilla+luciferase+reporter+gene+expression+in+living+mice.">Optical imaging of Renilla luciferase reporter gene expression in living mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 377-382 (2002).</li><li>Tisi, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+thermostable+firefly+luciferase.">Development of a thermostable firefly luciferase.</a> Analyica Chimica Acta. 457, 115-123 (2002).</li><li>Ray, P., Tsien, R., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+validation+of+improved+triple+fusion+reporter+gene+vectors+for+molecular+imaging+of+living+subjects.">Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects.</a> Cancer Res. 67, 3085-3093 (2007).</li><li>Cao, F., 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=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> . Circulation. 113, 1005-1014 (2006).</li><li>Cao, F., 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=Molecular+Imaging+of+Embryonic+Stem+Cell+Misbehavior+and+Suicide+Gene+Ablation.">Molecular Imaging of Embryonic Stem Cell Misbehavior and Suicide Gene Ablation.</a> Cloning and Stem Cells. 9, 107-117 (2007).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Wu, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+reporter+genes+for+molecular+imaging+of+transplanted+embryonic+stem+cells.">Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells.</a> Proteomics. 6, 6234-6249 (2006).</li><li>Eiges, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Mohr, J. C., de Pablo, J. J., Palecek, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+human+embryonic+stem+cells:+Small+and+macromolecule+loading+and+DNA+transfection.">Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection.</a> Biotechnol Prog. 22, 825-834 (2006).</li><li>Siemen, H., 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=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Lakshmipathy, U., 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=Efficient+transfection+of+embryonic+and+adult+stem+cells.">Efficient transfection of embryonic and adult stem cells.</a> Stem Cells. 22, 531-543 (2004).</li><li>Ma, Y., Ramezani, A., Lewis, R., Hawley, R. G., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Level+Sustained+Transgene+Expression+in+Human+Embryonic+Stem+Cells+Using+Lentiviral+Vectors.">High-Level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors.</a> Stem Cells. 21, 111-117 (2003).</li><li>Menendez, P., Wang, L., Chadwick, K., Li, L., Bhatia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+transduction+of+hematopoietic+cells+differentiated+from+human+embryonic+stem+cell-derived+CD45(neg)PFV+hemogenic+precursors.">Retroviral transduction of hematopoietic cells differentiated from human embryonic stem cell-derived CD45(neg)PFV hemogenic precursors.</a> Molecular therapy. 10, 1109-1120 (2004).</li><li>Thyagarajan, B., 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=Creation+of+engineered+human+embryonic+stem+cell+lines+using+phiC31+integrase.">Creation of engineered human embryonic stem cell lines using phiC31 integrase.</a> Stem cells. 26, 119-126 (2008).</li><li>Liew, C. -. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transient and Stable Transgene Expression in Human Embryonic Stem Cell. , .</li></ol>

PETやMRIなどの他のモダリティに比べて、生物発光は、限られた空間分解能と放出された光子の比較的弱いエネルギー（2-3 eV）のために少なく組織の浸透があり、これらの理由から、それはこれまで大規模な動物には適用されていない。しかし、生物発光は、小動物の追跡生体の幹細胞のためのそれは非常に望ましいこと、低コスト、高スループット、および非侵襲性という利点があります。 PETや蛍光構造?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BD Matrigel&#x2122; Basement Membrane Matrix</td>
<td>Growth factor reduced (optional: phenol-red free)</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-Luciferin Firefly, potassium salt</td>
<td>&nbsp;</td>
<td>Biosynth AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td>
</tr>
<tr>
<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6-well tissue culture-treated plates</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>92006</td>
<td>&nbsp;</td>		</tbody>
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in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

ヒト胚性幹細胞（ヒトES）の発見は劇的に再生医療に興味のある医学者が利用できるツールが増えています。しかし、生体に、ヒトES細胞から分化したヒトES細胞、および細胞の直接注入は、これまで顕著な細胞死、奇形腫形成、および宿主の免疫拒絶反応によって妨げられている。移植後のin vivoでヒトES細胞の行動に理解することは新たなイメージング技術は、長手方向にhESCのローカライズ、増殖、および生存率を監視する必要があります。分子イメージングは​​、調査官の数日、数週間、さらには数ヶ月にわたってin vivoでの細胞増殖に追跡するための、ハイスループットで安価、かつ高感度な手段を与えている。この進歩は著しくhESCの生着、増殖、そして生きている被験者におけるテラトーマ形成の時空間動態の理解を増加している。 分子イメージングにおける大きな進歩は、生体内でのマルチモダリティ画像処理プラットフォームへの分子細胞生物学から非侵襲的なレポーター遺伝子アッセイの拡張となっています。これらのレポーター遺伝子は、宿主細胞の転写機構を利用する人工プロモーターとエンハンサーの制御下、ベクトルおよび非ベクトルの様々な方法を用いて細胞に導入されています。一度セルに、レポーター遺伝子が使用されるプロモーターの種類に応じて、特定の生物学的または細胞の条件の下で恒常的にまたはいずれか一方のみ転写することができます。生理活性タンパク質へのレポーター遺伝子の転写と翻訳は、その後、D -ルシフェリンのような信号を発生するプローブを使用して機密情報、非侵襲的計測機器（例えば、CCDカメラ）で検出される。 蛍光イメージングのために必要とされるin vivoで幹細胞を追跡するために、興奮性、光の必要性を避けるために、生物発光レポーター遺伝子イメージングシステムは、発光を誘導するだけ外因的に投与プローブが必要です。ホタルPhotinusスピラリスから派生したホタルルシフェラーゼは、光学活性代謝物、オキシルシフェリンにD -ルシフェリンを触媒する酵素をコードしている。光学活性を、外部のCCDカメラで監視することができます。安定してレポーターを運ぶ細胞はその染色体DNA内に構築形質レポーターがin vivoでのヒトES細胞の生存や増殖の長手方向の監視を可能にする、娘細胞にDNAを構築渡します。レポーター遺伝子産物の発現は、信号の生成に必要となるためさらに、唯一の現実的親と娘細胞は、生物発光のシグナルを作成します。アポトーシスまたは死細胞はしません。 このビデオでは、生物発光イメージングによる追跡幹細胞の増殖と奇形腫形成に必要な特定の材料および方法について説明する。

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

幹細胞治療への関心の高まりとともに、分子イメージング技術は、移植後の幹細胞の挙動のモニタリングに最適です。ルシフェラーゼレポーター遺伝子は、細胞の生存、場所、およびin vivoでの増殖の非侵襲的、反復的な評価を可能にした。このビデオでは、生きたマウスでのヒトES細胞の増殖を追跡する方法を説明します。

in vitroおよびヒト胚性幹細胞のin vivoでの生物発光レポーター遺伝子イメージングの

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

in-vitro-vivo-bioluminescence-reporter-gene-imaging-human-embryonic

Research

JoVE Journal

Biology

21.7K ビュー.  Stanford University School of Medicine. 幹細胞治療への関心の高まりとともに、分子イメージング技術は、移植後の幹細胞の挙動のモニタリングに最適です。ルシフェラーゼレポーター遺伝子は、細胞の生存、場所、およびin vivoでの増殖の非侵襲的、反復的な評価を可能にした。このビデオでは、生きたマウスでのヒトES細胞の増殖を追跡する方法を説明します。

ビデオ: In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

生物学

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

ヒト胚性幹細胞のミトコンドリア複合体活性の状態調査

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

磁気共鳴イメージングのためのヒト胚性幹細胞のin vitroでラベリングの

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

ヒト胚性幹細胞を凍結融解

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

ヒト胚性幹細胞のコロニーの成長のビデオバイオインフォマティクス解析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

オリゴデンドロサイト前駆体に胚性幹細胞の分化

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...