装置<ul><li>組織培養インキュベーター、37℃、5％CO 2 </li><li>遠心</li><li> micropipettorsのセット（100、100μL） </li><li>水浴37℃ </li><li>水浴90℃の蒸気の水の源になるために</li><li>位相差顕微鏡</li></ul> FIRST STEP コルセミドで細胞を処理するこの手順では、20％ノックアウト血清代替（KSR、ギブコ社）と8ng/mLのを補充マイトマイシンC（シグマ）で不活化とDMEM/F12（Invitrogen）中で維持マウス胚線維芽細胞（MEF）上に培養したヒトES細胞のラインH9を使用線維芽細胞増殖因子（FGF - 2、R＆D）。 分裂中期スプレッドの多数を得るために、それを多くの分裂細胞が存在するで高い分裂インデックスを持つ文化を使用することをお勧めします。 <ol><li>インキュベーターから培養フラスコを取ると、層流で、0,1μg/ mLの最終濃度でコルセミド追加。 </li><li>インキュベーターにフラスコを返し、阻害剤は、染色体がうまく凝縮されており、顕微鏡下で容易に可視化できる中期、上に有糸分裂停止を引き起こす可能性があるので、3時間を待つ。 </li></ol>細胞を固定<ol start="3"><li>培地を取り出し、細胞の表面をカバーするのに十分トリプシン/ EDTA、0.05％追加。細胞は室温で5分間インキュベートし、血清を含む培養培地を追加したり、単に細胞に血清添加することによりどちらかの酵素を不活性化することができます。 注：単一の細胞懸濁液は、良好な分裂中期スプレッドを取得するために達成されていることを確認することが重要です。 </li><li> 15 ml遠心チューブと5分は122 gで遠心して細胞を移す。 </li><li>約200 mLを残し、上清を捨て、そして、ビデオに示すようにチューブを、タップして穏やかにペレットを再懸濁します。細胞がよく解離されるので、ソリューション内の任意の塊を視覚化できないことを確認してください。 </li><li>次に、低張液5 mL（塩化カリウム75mmの）37に予め温めておいた° C、ゆっくりと、以下の説明のように管壁によって。を追加する必要があります。最初だけでその後低張液で細胞を混ぜ、残りの2 mlを加え37℃で保温℃で15分間に水平位置にチューブを反転、3 mlを加える。 注：この時点で、低張液は細胞のボリューム上の増加を引き起こし、染色体を解くのに役立ちます。インキュベーションの時間は、良好な染色体のスプレッドを獲得することが重要です。タイミングが長すぎる場合、細胞膜には早すぎると染色体が失われて破裂することがあります。短すぎる場合、細胞膜が中断されないことがありますので、それは染色体のスプレッドを得ることが困難な場合があります。 </li><li>固定液（メタノール/氷酢酸3:1）の3滴を追加、休憩なしで5分間、122 グラムの細胞と遠心機で固定溶液を混合して軽くチューブを反転させる。 注：固定溶液をたててください。 注：材料の損失を防止する、細胞の減少を避けるために、休憩なしで遠心分離を使用することが重要です。 </li><li>約200 mLを残し、上清を捨て、そしてチューブの底を軽くたたくことによって静かにペレットを再懸濁します。 注記：ここでも、細胞がよく解離されるので、ソリューション内の任意の塊を視覚化できないことを確認してください。 </li><li>もしチューブを回転しながら、管壁が徐々に、固定液の最初の3 mLを加え。単に細胞を混合し、細胞をダウンさせるために管壁で固定溶液中より2 mLを追加するには、水平位置にチューブを反転。 4℃で一晩株式。 注：固定された細胞は4℃ヶ月間固定液に保存することができます℃のその膨潤状態で細胞を保護する以外に、固定液は脂質と変性蛋白質を削除します。これらのイベントは、結果として、染色体は容易に拡散させる、細胞膜が壊れやすいようにと。 </li></ol>スライドガラスのクリーニングあなたの染色体スプレッドを作り始める前にスライドが適切に洗浄されているかあなたには、いくつかの核とも中期を失う可能性があることを確認してください。あなたがどんなハイブリダイゼーション技術9試してみたい場合は、このステップでは、同様に重要です。 <ol><li>場所は、室温で少なくとも3時間のために6M塩酸を入れたビーカーにスライドします。 </li><li>この時間の後、10分間水道水を流してのスライドを洗う。 </li><li>スライドを蒸留水ですすぎ、彼らは、室温で乾燥させます。 </li><li>今スライドを96％エタノール中に保存され、使用の直前に、糸くずを乾燥させることができる。 </li></ol>注：in situハイブリダイゼーション法では、スライドを96％エタノールに2日以上保存することができます。 SECOND STEP 細胞を洗浄<ol start="10"><li>次の日に、休憩なしで5分間、122 グラムで細胞を遠心分離。 </li><li> D約200mlを残して上清をiscard。軽くチューブを回転させながら、チューブは、チューブの壁が徐々に、新鮮な固定液を5ml（メタノール/氷酢酸3:1）を追加するフリックでペレットを再懸濁します。この洗浄は3回の洗浄の合計を2回以上繰り返します。 注：固定溶液をたててください。 </li></ol>スライドの準備<ol start="12"><li>最後の遠心分離工程において（図1参照）細胞ペレットを均一化しスライドに細胞の十分な密度を得るために解決策の十分な量を残す。 </li><li>非常に近いと表面に平行にピペットを移動することで、清潔で乾燥したスライド上に細胞懸濁液の20〜30μLを加える。固定液が蒸発するように、スライドの表面が粗くなります。 </li><li>すぐに、細胞が爆破させるために30秒間お湯の蒸気（90 °）に、表向き、スライドを公開します。 注：この時点では、一緒に水で拡散染色体を刺激し増加酢酸濃​​度、結果として固定溶液が蒸発するからメタノール、。 </li><li>濃度だけでなく、細胞の分布は（図1参照）良いかどうかだけをチェックする位相差顕微鏡で見てください。 </li><li>現在のスライドは、G -バンディング（ギムザ）、FISH、SKYやCGHなどの細胞遺伝学的アプローチによる染色体のセットをチェックするために使用する準備が整いました。 </li></ol>重要なヒント分析するに十分な中期の方法を選択ラウンドと分離された分裂中期（図2D）を選択するには、2つまたはそれ以上の広がり紛れ込まない起動染色体によって生成された染色体の消失によって生成された染色体のゲインとして偽aneuploidiesを考慮して回避することができます。ので、いくつかの染色体は、それら（図2Aおよび2B）により隠れている可能性がありますので、原子核の近くに分裂中期の選択を避ける。ほかに、ラウンド分裂中期を探して、任意の染色体が分裂中期（図2C）から分離されていることを確認してください。 フィーダー細胞とヒトES細胞の核型 G -バンディングとSKYの分析のために、ヒトES細胞培養におけるフィーダー細胞の存在は、フィーダー細胞が分裂状態下にないため、結果を妨げることはありません（マイトマイシンCまたは照射によって不活性化）と中期の人口の一部にはなりません。しかし、間期核のFISH法の応用のために、それはFISH解析のために、トリプシン処理する前に手動でコロニーを収穫によってフィーダー細胞からヒトES細胞の集団を分離することが好ましい。 <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="図1" /> 図1：どのようにスライドの適切な細胞密度を知っている。 10倍の目標の位相差顕微鏡の画像。 （A）細胞密度が高すぎる。矢印は核の近すぎる広がる中期を指している。 （B）良好な細胞密度とスライド。円は、核または他の分裂中期から分離分裂中期スプレッドを示しています。してください<a href="https://www.jove.com/files/ftp_upload/1512/1512fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="図2" /> 図2：どのように分析するに十分な中期を選択する。染色体はDAPIと100xの目的での蛍光顕微鏡によって得られた画像で染色されています。 （A）（B）核の近くに分裂中期（矢印で示される）（C）を避け、現在の染色体は、（丸）で区切られたことが丸くなっていない分裂中期。 （D）ラウンド中期分析する良い中期です。してください<a href="https://www.jove.com/files/ftp_upload/1512/1512fig2.jpg">ここをクリックして</a>図2の拡大バージョンを参照すること。

<ol>
	<li>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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1145 (1998).</li><li>Draper, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+gain+of+chromosomes+17q+and+12+in+cultured+human+embryonic+stem+cells.">Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.</a> Nat Biotechnol. 22 (1), 53-53 (2004).</li><li>Maitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+alterations+in+cultured+human+embryonic+stem+cells.">Genomic alterations in cultured human embryonic stem cells.</a> Nat Genet. 37 (10), 1099-1099 (2005).</li><li>Mitalipova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-19 (2005).</li><li>Buzzard, J. J., Gough, N. M., Crook, J. M., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype+of+human+ES+cells+during+extended+culture.">Karyotype of human ES cells during extended culture.</a> Nat Biotechnol. 22 (4), 381-381 (2004).</li><li>Caisander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+integrity+maintained+in+five+human+embryonic+stem+cell+lines+after+prolonged+in+vitro+culture.">Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.</a> Chromosome Res. 14 (2), 131-131 (2006).</li><li>Imreh, 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=In+vitro+culture+conditions+favoring+selection+of+chromosomal+abnormalities+in+human+ES+cells.">In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.</a> J Cell Biochem. 99 (2), 508-508 (2006).</li><li>Robin-Wesselschmidt, J. L., Loring, J. F., Robin-Wesselschmidt, J. L., Robin-Wesselschmidt, S. c. h. w. a. r. t. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Stem Cell Manual. , 59-59 (2007).</li><li>Rooney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cytogenetics. , (2001).</li><li>Heslop-Harrison, P., Schwarzacher, T., Heslop-Harrison, P., Schwarzacher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical in situ hybridization. , 51-51 (2000).</li></ol>

The authors have nothing to disclose.

染色体スプレッドの準備は、G -バンディング、およびそのようなFISH、SKYやCGHなどのより高度なテクニックとして日常的に技術による胚性幹細胞の遺伝的状態を正常に解析するための重要なステップです。 この手順は、細胞周期の長さに依存するコルセミドインキュベーション、期間を変化させることで、多くの細胞型に適用することができます。胚様体（EB）のために?...

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<td>15 ml centrifuge tube</td>
<td>&nbsp;</td>
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<td>Colcemid Karyo MAX</td>
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<td>Glacial acetic acid</td>
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<td>100</td>
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chromosomal spread preparation of human embryonic stem cells for karyotyping

ヒト胚性幹細胞（ヒトES細胞）安定した二倍体の核型1を提示することが示されているが、多くの研究は、培養条件に応じて、彼らはそのような全体または染色体の部品の追加などの染色体異常を取得する傾向になることを報告している。確かに、長期培養の間に、核型の変化はときに酵素的または化学的解離が観察される継代のためのコロニーのマニュアル解剖が安定核型5を保持しながら、2,3,4が使用されています。加えて、フィーダー細胞の除去など環境の変化はまた、ヒトES細胞3,6の遺伝学的完全性が損なわれるようです。一度染色体変異が細胞生理に影響を与える可能性、in vitroでのヒトES細胞の遺伝的整合性の特性は、胚発生の研究と薬物検査に不可欠なツールとして重要な考慮hESCのです。さらに、将来の治療目的のために染色体の変化が頻繁に発癌に関連付けられているように大変な問題です。 ここでは、G -バンディング、FISH、SKYやCGHの技法7,8で設定された染色体のその後の分析のために高品質の染色体のスプレッドを得るために簡単で便利な方法を示しています。我々は、転座とaneuploidiesの外観をモニターするために5継代の間隔で定期的に染色体の状態を確認することをお勧めしますプリシラブリットとラファエラサルトルは、紙にも同様に貢献した。

Watch this Scientific Journal Video about Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping at JoVE.com

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

核型分析は、広く遺伝子変異を検出するために使用されるシンプルかつ有用な技術である。ここでは、文化の中で維持、これらの細胞の染色体の状態を監視するためのヒト胚性幹細胞の染色体の広がりの準備のためのステップのプロトコルバイステップで説明します。

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 ...

chromosomal-spread-preparation-human-embryonic-stem-cells-for

Research

JoVE Journal

Biology

48.5K ビュー.  Federal University of Rio De Janeiro-UFRJ. 核型分析は、広く遺伝子変異を検出するために使用されるシンプルかつ有用な技術である。ここでは、文化の中で維持、これらの細胞の染色体の状態を監視するためのヒト胚性幹細胞の染色体の広がりの準備のためのステップのプロトコルバイステップで説明します。

ビデオ: Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

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 ...

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 ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.