著者はアンドレアアクステル聡伊藤、MD、ジェフVelotta、MD、グラントホイト、ロバートC.ロビンス、MD、金玉、M​​D、ティムドイル博士、および技術支援のためのスタンフォード大学小動物のイメージングコアを感謝。また、作者は獣医機器のサポートのためにAMビックフォード社に感謝。この研究は国立衛生研究所（R01 HL - 75774、R01 CA098303、R21 HL085743、1K12 HL087746）、カリフォルニア大学のカリフォルニア州のたばこ関連病研究プログラム（15IT - 0257と1514RT - 0169）からの研究補助金によって支えられて、および再生医療（RS1 - 00183）のためのカリフォルニア工科大学。 NHは、アメリカ心臓協会からのフェローシップでサポートされています。心臓協会ができます。

1。内皮細胞へのマウスES細胞の分化<ol><li>内皮細胞にES細胞を区別するためのプロトコルは別の場所で記述し、このプロトコル2,3の焦点ではありません。簡単に言えば、細胞は区別するために許可され、そしてそのようなCD31または血管内皮カドヘリン（VE -カドヘリン）のような内皮細胞マーカー陽性の細胞は、蛍光活性化細胞選別（FACS）によって精製されています。 </li></ol> 2。ダブル融合レポーター遺伝子とレンチウイルス形質導入の建設<ol><li>生物発光は、ホタルルシフェラーゼ（変動を確認）などのレポーター遺伝子を発現するように変更されている細胞を追跡するために使用することができます。このアプリケーションでは、我々の細胞は内部のユビキチンプロモーターの制御下に両方の変動を確認し、強化された緑色蛍光タンパク質（EGFP）融合遺伝子が含まれています。セルを変更するために、バイオセーフティ基準と同様に増加した形質導入効率を達成するために重要な最適化された遺伝的要素を含むトランスジーンレンチウイルスベクターは、変動を確認- EGFP融合レポーター遺伝子を運ぶ私たちの研究室で開発PFU - FGのベクトルが安定するために使用することができます。分化後の細胞を伝達する。この融合構築物は、移植細胞の生物発光と蛍光の追跡の両方が有効になります。レポーター遺伝子を発現する標識された細胞のウイルス粒子、伝達し、生産を生成するための手順は、他の4説明されています。 </li></ol> 3。虚血後肢にESC -由来の内皮細胞の移植<ol><li>細胞移植のための後肢虚血を受けているマウスを用意することで手順を開始します。これを行うには、1L/minの流量で100％酸素でイソフルラン1〜3％を含む麻酔導入チャンバーにマウスを置きます。 </li><li>それは外的刺激に反応しなくなるまで誘導チャンバ内でマウスを残す。その後、誘導室から動物を削除します。 </li><li>その後、手術台の上に仰臥位に動物を配置し、1L/minの流量で100％酸素でイソフルラン1〜3％連続流に接続してください。 </li><li> three交互betadineとアルコールスクラブと後肢の皮膚を拭いてください。 </li><li>肌がきれいにされると、（PBS）リン酸緩衝生理食塩水の30μLを、100万ESC -由来の内皮細胞を得る。 28ゲージの針に、これらの細胞をロードする。 </li><li>細胞の準備が整うと、静かに持ち上げて、より良い腓腹筋の場所を視覚化するために後肢を拡張。足が拡張されている間、基本的な筋肉に皮膚を通して針を挿入する。骨に接近しないように注意してください。静かにゆっくりと腓腹筋に30μLの細胞混合物を注入する。筋肉内注射の場合、30μLの注入可能な量の限界に近いです。我々の経験で、彼らは注射針での損失量を排除する、それ故、28ゲージのインスリン注射が好ましい。 </li><li>注入が完了すると、回復のケージにマウスを返し、目を覚ましになるまで継続的に監視する。動物は、数時間で回復し、移植細胞のin vivo生物発光イメージングを続行することができます。 </li></ol> 4。 in vivoでのESC -由来の内皮細胞の生物発光イメージング<ol><li>移植ESC由来endothelials細胞は内部のユビキチンプロモーターの制御下で変動を確認し、EGFPの融合遺伝子の両方を発現するように変更されました。従って、生物発光は、虚血後肢内の細胞を追跡するために使用することができます。 </li><li>このステップを開始するには、生物発光イメージングシステムとリビングの画像取得ソフトウエアをオンにします。その後、収集システムを初期化し、視野の大きさを指定します。 </li><li>背景放射を吸収するために、イメージングボックスの上に横に、場所黒のマット紙。 </li><li>イメージングボックスの準備が整ったら、1L/minの出力で酸素のイソフルラン1〜3％を含む麻酔導入チャンバーにマウスを置きます。それは外的刺激に反応しなくなるまで誘導チャンバ内でマウスを残す。その後、誘導室から動物を削除します。 </li><li>必要に応じて電気カミソリを使用して、両方の後肢から毛を取り除く。 </li><li>腹膜に体重1g当たり、D -ルシフェリンの10μLを注入する。 D -ルシフェリンをPBS中の15 mg / mLのフィルタリングストック溶液に予め用意されています。 </li><li>ルシフェリンが注入されると、イソフルランの連続的な流れに接続して仰臥位で、黒い紙、上の画像ボックスに動物を配置。 </li><li>イメージングボックスに動物を配置し、イソフルランに接続する。 </li><li>画像が飽和されていない最適な露光時間を決定するために、10〜60秒のために画像を取得するために始める。画像が飽和状態になった場合、露光時間を減らす。生物発光シグナルは非常に弱い場合は、露出時間を増やします。 </li><li> Usinグラム最適な露光時間は、信号が最大としてフェードに達するまで毎に1-3分の画像を取得し続けます。買収が完了したら、ファイルを保存します。 </li><li>データを分析するために、注射部位をカバーする観測領域（ROI）を選択します。ネガティブコントロールとして、同様のROIは、非手術足のために選択することができます。ソフトウェアを使用して、各時点に対してROIのための光子/秒/ cm 2で /ステラジアン（P / S / cm 2で / SR）の単位で表現されている合計の輝きを、測定する。最大値は、最終的なデータで使用する必要があります。データはまた、将来の使用のためのExcelスプレッドシートにエクスポートすることができます。 </li><li>一度すべてのデータが取得されている、回復のケージに動物を戻すと、動物の目を覚ましまで継続的に監視する。 </li><li>時間をかけて細胞を追跡するために、この手順を繰り返します。 </li><li>希望する時点で、動物は、組織機能の評価のために安楽死することができます。 </li></ol> 5。代表的な結果<img src="/files/ftp_upload/old/1034_2009_1_22_4/figure 1.png" alt="図1" width="600" height="600" />左の虚血後肢における移植細胞の代表的な生物発光画像を図1に示されています。生物発光の買収時には、強度は時間とともに増加し、時間の経過中に得られた最大値を最終値として報告されるべきである。

<ol>
	<li>Niiyama, H., Huang, N. F., Rollins, M., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> JoVE. , (2008).</li><li>Levenberg, S., Golub, J. S., Amit, M., Itsakovitz-Eldor, J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+derived+from+human+embryonic+stem+cells.">Endothelial cells derived from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 4391-4396 (2002).</li><li>Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk1-positive+cells+derived+from+embryonic+stem+cells+serve+as+vascular+progenitors.">Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.</a> Nature. 408, 926-926 (2000).</li><li>De, A., Yaghoubi, S. 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=Applications+of+lentiviral+vectors+in+noninvasive+molecular+imaging.">Applications of lentiviral vectors in noninvasive molecular imaging.</a> Methods Mol Biol. 433, 177-202 (2008).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+laboratory+mouse.">The anatomy of the laboratory mouse.</a> , (1976).</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 Med. 4, 245-247 (1998).</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>Huang, N. F., Lee, R. J., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+regulation+of+stem+cells+and+progenitor+cells:+potential+for+cardiovascular+tissue+engineering.">Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering.</a> Tissue Eng. 13, 1809-1823 (2007).</li><li>Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X., Weissman, I. L., Gambhir, S. S., 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=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, 1005-1114 (2006).</li><li>Wilson, K., Yu, J., Lee, A., 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=In+vitro+and+in+vivo+bioluminescence+reporter+gene+imaging+of+human+embryonic+stem+cells.">In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.</a> J Vis Exp. , (2008).</li></ol>

ESCは、ために分化し、血管内皮細胞を含むすべての3つの胚芽層を、前記の細胞系譜を生じさせる能力の彼らの可塑性の組織の虚血の治療において有望な細胞源です。 ES細胞に関連する倫​​理的な問題を克服するために、誘導多能性幹細胞（性IPSC）倫理的な問題を克服する代替の多能性幹細胞の源とすることができる。 ESCのほかに、そのような内皮前駆細胞（EPC）と造血幹細胞（HSC）のような成体幹細胞...

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		<th>Comment</th>
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<td>Surgical tools</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Syringe needle</td>
<td>Tool</td>
<td>BD</td>
<td>&nbsp;</td>
<td>28G insulin syringe is preferred</td>
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<td>Phosphate Buffered Saline </td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
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<td>D-luciferin</td>
<td>Reagent</td>
<td>Biosynth International, Inc</td>
<td>&nbsp;</td>
<td>Prepare D-luciferin in advance into filtered stock solutions of 15 mg/mL in PBS</td>
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<td>IVIS 200 Bioluminescence imaging system and acquisition software </td>
<td>Equipment</td>
<td>Xenogen Corporation</td>
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embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia

脚と足に酸素化血液と栄養分を供給する末梢動脈の狭窄から末梢動脈疾患（PAD）の結果は、この病理は、このような間欠性跛行（歩くと痛み）、痛みを伴う虚血性潰瘍、あるいは四肢を脅かす壊疽などの症状を引き起こします。これは、一般的に血管内皮、すべての血液やリンパ管の管腔表面を投資する内皮細胞の単層は、血管の恒常性と血管再生で主要な役割を果たしていると考えられている。その結果、内皮細胞の幹細胞ベースの再生には、PADを治療するための有望なアプローチがあります。 このビデオでは、我々は胚性幹細胞（ESC）由来の生物発光イメージングによるホーミング細胞と生存の非侵襲的な追跡が続くPADのモデルとして一方的hindimbの虚血の治療のための内皮細胞の移植を示しています。細胞送達およびイメージングのための特定の材料および手順について説明する。このプロトコルは、新山らによる後肢虚血の誘導を記述する別のパブリケーションに従います。1

Watch this Scientific Journal Video about Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia at JoVE.com

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

胚性幹細胞の配信のための外科的処置は、虚血後肢への細胞由来の内皮細胞は、生物発光イメージングによる非侵襲的な追跡と、示されている。

後肢虚血の治療のための胚性幹細胞由来血管内皮細胞

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

embryonic-stem-cell-derived-endothelial-cells-for-treatment-hindlimb

Research

JoVE Journal

Biology

11.7K ビュー.  Stanford University. 胚性幹細胞の配信のための外科的処置は、虚血後肢への細胞由来の内皮細胞は、生物発光イメージングによる非侵襲的な追跡と、示されている。

ビデオ: Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

ヒト胚性幹（ES）細胞の増殖

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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 ...

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 ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

後脚虚血症のマウスモデル

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

染色体分析のためのヒト胚性幹細胞の染色体スプレッドの準備

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

運動ニューロンにマウス胚性幹細胞の効率的な分化

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...