1。メンテナンス：顕微鏡下で観察する文化<ol><li> 37℃のインキュベータからヒトES細胞の一つのプレートを取り外し、顕微鏡にもたらす。 </li><li>全体的な文化の質を評価するために、2倍または5倍の倍率を使用して、低消費電力でのコロニーを観察します。通常の培地の色、目に見える汚れ、全体のコロニーの大きさ、質および密度の欠如、および、他の観測：次の点に注意してください。 </li><li>培地の色の変化を観察する。 pHの変化とヒトES細胞との一晩のインキュベーション後の培地中の栄養素の枯渇のために、媒体が&quot;わら&quot;色に赤の色相から変わります。培地が紫表示された場合は、基本的なpHのシフトが発生しました。媒体は非常に黄色が表示された場合、媒体が酸性化している。非常に黄色の培地はよくまたは汚染に非常に過密なコロニーの結果である可能性があります。培地は、常に明確に表示されます。培地中の細胞残渣の一定のレベルは正常ですが、それは曇って見えることはありません。細胞残渣の高レベルが観察されている場合は、文化は健全ではないと汚染されることがあります。 </li><li>文化は保守や継代を必要としない場合、それらは（セクション2を参照）に供給できるように生物学的安全キャビネットに撮影することができます。 </li><li>培養物が約10％のコロニーを差別化よりも多く含まれている場合は、セルを手動で差別化された領域（セクション3を参照）を削除して&quot;クリーンアップ&quot;する必要があります。 </li><li>文化は大規模または密集コロニーが含まれている、またはMEFフィーダー層以上12日間経過している場合、セルは（セクション4を参照）継代してください。 </li></ol> 2。摂食hES細胞<ol><li>無菌生物学的安全キャビネット内で、培養皿の蓋を外し、使用済み培地を吸引除去する。ピペットの先端が直接井戸内よりも他の板の任意の部分に触れないようにしてください。汚染の心配がある場合は、新しい、滅菌ピペットに変更。 </li><li>滅菌ガラスピペットを用いて、各ウェルに加温し、ヒトES細胞培養培地の適切な量を追加します。 6ウェルプレートでの培養のために、ウェルあたり2.5mlの培地を加える。 </li><li> 37℃、5％CO 2で推移するインキュベーターにプレートを返します。 </li></ol> 3。ヒトES細胞培養物から差別を削除する<ol><li>成形アルコールバーナーの制御された炎上のヒントでピッキングツールに9インチのガラスパスツールピペットを変換します。ピペットの先端が汚染を防ぐために密封し、培養皿のプラスチックに傷をつけないように丸められることを確認してください。生物学的安全キャビネットまたはピッキングの筐体にUV光源を使用して、使用前にピッキングツールを滅菌する。 </li><li>差別化の領域を削除します。分化は、多くの場合、通常のエッジに沿って、または中央に孤立したスポットとして、ヒトES細胞のコロニーの一部のみで発生します。コロニーのセクションのみが差別されている場合は、唯一の差別化された領域を削除する必要があります。差別は、しばしば&quot;スティッキー&quot;シートでプレートをオフに持ち上げることができる、そしてそれはピッキングツールの終わりに植民地を通る線を描画することにより、コロニーの残りの部分から分化した細胞を最初に分離すると便利です。 </li><li>コロニーの部分が分離されると、静かに不要な細胞を分離するためにプレートに沿って、ピッキングツールを滑ります。この過程で植民地の間にあまりにも多くのMEFフィーダー層のを削り取るように注意してください。 </li><li>分化した細胞のすべてが、プレート（と培地中に浮遊）から削除すると、生物学的安全キャビネットに文化を返します。分化細胞を含む培地を吸引除去し、新鮮なヒトES細胞培養培地と交換してください。 </li></ol> 4。継代ヒトES細胞酵素のインキュベーション<ol><li>無菌生物学的安全キャビネット内で、6ウェル培養皿の蓋を取り外し、継代される井戸から使用済み培地を吸引除去する。ピペットの先端が直接井戸内よりも他の板の任意の部分に触れないようにしてください。 </li><li>滅菌ガラスピペットを用いて、継代される各ウェルに加温し、コラゲナーゼIVの1 mLを加える。 </li><li> 5分間37℃のインキュベーターにプレートを返します。 </li></ol>ヒトES細胞を掻き取りとプーリング<ol start="4"><li>コラゲナーゼで細胞をインキュベートした後、顕微鏡下でコロニーを観察。酵素は、コロニーの端で微妙だが目に見える変化を引き起こす必要があります。 </li><li>生物学的安全キャビネット内で、静かに各ウェルから酵素を吸引し、2.0 mLをヒトES細胞培養用培地と交換してください。 </li><li>先端わずかに向かって培養プレートと5mLのガラスピペットで最初のウェルに2.0 mlの培地を取る。徐々にメディアのリリース中にプレートの底にピペットに対して垂直に保持する、静かにも渡っピペットチップを滑ります。 </li><li>こするとpipettiを繰り返しますngの動き3-4倍は植民地のすべてまでもから削除されています。ピースの小さすぎるにコロニーを破壊を避けるために穏やかにピペッティング。細胞が最初に井戸から削除されているときは、よくで内容のままにしても次の細胞を掻き始める。 </li><li>継代される井戸のすべてが削り取られて、プール滅菌15 mLコニカルチューブの各ウェルからコロニーの部分を含有する培地を。 </li><li>各ウェルにヒトES細胞培養の培地1mLを加えることによって掻き井戸をすすぐ。これはリンスとコニカルチューブに移す集める。 </li><li>ピペットで静かにチューブ内の細胞が目的のサイズにコロニーの部分を分割する。 </li><li> 200 × gで5分間、細胞を遠心分離</li></ol> hES細胞のセルをめっき<ol start="12"><li>ヒトES細胞は遠心分離している一方で、新しい6ウェルMEFフィーダープレートを準備。細胞株、新しい継代数、および通過の日付：適切なhES細胞の情報をプレートにラベルを付けます。井戸からMEFの培地を吸引除去し、各ウェルに1.0mLのPBSを加える。ゆっくりと渦は井戸の周りにバッファとPBSの洗浄を吸引除去する。各ウェルに1.5 mLのヒトES細胞培養培地を追加し、プレートを横に置きます。 </li><li>ヒトES細胞を遠心分離が終了したら、生物学的安全キャビネットにチューブを戻す。ゆるく充填された細胞ペレットを乱さないように注意しながら、上清を吸引除去する。 </li><li>ウェル当たり1 mLのヒトES細胞培養の培地は、被めっきするための十分な培地でペレットに細胞を再懸濁します。 6新しい井戸に分割するときは、6 mLの培地でペレットを再懸濁します。 </li><li>優しくして均等にMEFフィーダープレートの各ウェル準備に細胞懸濁液1 mLを加える。 </li><li> 37℃のインキュベーターにプレートを置き、慎重に均等によく中の細胞を配布する側にバックアップするために前方にプレート、そして側面をスライドさせます。 </li><li>細胞は37℃で一晩にアタッチすることができます。 </li></ol> 5。代表的な結果： 未分化hES細胞は小さく、密集している、と通常は細胞外より大きく、より広がりで構成されています。分化は、コロニー（図1A）内にまたはコロニー（図3B）の間に発生する可能性があります。 異なるmorhopologiesは、顕微鏡下で低倍率で見ることができます。図2aに示すように良好な細胞形態は、単層で成長小さな、すし詰め状態の細胞が含まれています。細胞は分化がほとんどで、清潔で、定義されたエッジを持つ必要があります。図2Bに示すように、細胞は継代のための準備ができて、そして図2Cに示すように細胞が過密している。 <img src="/files/ftp_upload/1427/1427fig1.jpg" alt="図1" /> 図1。ヒトES培養における分化。（）コロニーの間にコロニー（B）分化の分化。 <img src="/files/ftp_upload/1427/1427fig2.jpg" alt="図2" /> 図2。ヒトES文化に見られる形態 。 （A）良好な細胞の形態（B）（C）過密状態の細胞を継代のための準備ができているヒトES細胞。

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique. , (2005).</li></ol>

無菌性は、ヒトES細胞を扱うすべての回で維持する必要があります。生物学的安全キャビネットと使用前に全ての機械を洗浄し、滅菌する。すべての試薬は使用前に0.22μmの孔径のフィルターを使用してフィルタ処理する必要があります。ヒトES細胞培養でantiboioticsの使用は必要ではなく、避けるべきです。 別々の環境は、指定されたピッキングステーションとして設定する必要があります。ステーションのための無菌エンクロージャはUV光源、層流フード、または生物学的安全キャビネットを持つ静的エンクロージャ（PCRなどのワークステーションなど）することができます。ピッキングステーション内部の解剖顕微鏡は、差別化の領域が削除されるコロニーを観察するために必要です。静的な筐体または生物学的安全キャビネットの前面のガラスパネルは、適切な通気と無菌性を損なうことなく、パネルを貫通する顕微鏡の接眼レンズを可能にするために変更する必要があります。 健康なヒトES細胞培養を維持するために、セルごとに4〜7日一般的に、最適な時点で継代する必要があります。この時点ではコロニーが彼らの最大サイズに達しているとマージし始めてされることがあります。マージされたコロニーは、培養中の分化の割合を増やすことができます。スプリット比は、一般的に植民地メッキ、膨張率、および培養条件の数に応じて、1:3および1:5の間になる。 Overplatedコロニーは（スプリット比が低すぎる）可能性が途中でマージし、彼らが最大サイズに達する前に継代する必要があります。 2週間以上古いですMEFフィーダー層上で培養物を、未分化の成長と増殖をサポートする新しいMEFをする上で継代する必要があります。 差別化が自然にヒトES細胞培養で行われるため、少量が期待されている。培養物は、適​​切な治療を受けていない場合は、頻繁にまたは過度の分化が発生する可能性があります。細胞が毎日供給する必要がある、通路との間の増殖は避けてください。常に新鮮な試薬を使用してください。メディア、MEFをと試薬のすべてが未分化hES細胞の増殖を防ぐために14日以内に使用する必要があります。このようなノックアウト血清代替、FBSおよびMEFを、などの試薬の新しいロットは、使用前にスクリーニングすべきである。継代する前に削除されていない任意の分化細胞が新しい文化に転送されることに注意してください。また、° C、可能な限り37℃で細胞を維持しようとします。細胞は細胞が長期間培養器からなる場合は加熱された顕微鏡のステージは非常に有益であることができる（顕微鏡のステージ上または生物学的安全キャビネット内など。）インキュベータの外に費やす時間を最小限に抑えます。 顕微鏡下でヒトES細胞培養を観察すると、最初のコロニーの全体的な品質と大きさを評価する低消費電力（2倍または5倍）対物レンズを用いて表示。潜在的に分化した細胞は、低消費電力で認められた場合には、より高い倍率の目標を（10X）を使用して差別化された形態を確認してください。 すぐに差別を除去した後、コロニーの縁がギザギザまたは破損して表示されることがあります。残りの未分化細胞が回復できるように新鮮な培地で一晩コロニーをインキュベートする。文化の分化の影響を受けず、これらの残りのコロニーでは、正常に増殖していきます。 可能であれば、低継代ヒトES細胞を用いた実験を始める。すべての主要な実験の前と後の細胞を核型。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF medium</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
</tr>
<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout&trade; Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>03-0002</td>
<td>Use at [4 ng/mL] final</td>
</tr>
<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-081</td>
<td>200 mM (100X) 
Use at [1X] final
</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>Without Ca2+ or Mg2+</td>
</tr>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>Type A, Porcine</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140675</td>
<td>For general culture</td>
</tr>
<tr>
<td>4-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
</tr>
<tr>
<td>5 mL glass pipets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
</tr>
<tr>
<td>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-20D</td>
<td>9&#x201D; glass</td>
</tr>		</tbody>
		</table>
		</div>

culture and maintenance of human embryonic stem cells  

ヒト胚性幹（ヒトES）細胞は健康な、未分化の文化を維持するために監視し、世話する必要があります。最低でも、文化が失われた栄養素を補充すると、不要な分化因子の自由な文化を維持するために完全な培地交換を行うことにより、毎日供給する必要があります。分化の少量が正常と幹細胞培養に期待ですが、文化は、日常的に手動で削除し、または差別化の領域を&quot;ピッキング&quot;でクリーンアップする必要があります。識別し、ヒトES細胞培養物から過剰な分化を削除すると、細胞の健康な個体群の維持に不可欠なテクニックです。

Watch this Scientific Journal Video about Culture and Maintenance of Human Embryonic Stem Cells at JoVE.com

Culture and Maintenance of Human Embryonic Stem Cells

このプロトコルは、健康的、未分化ヒト胚性幹（ES）細胞を維持する方法を示します。

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 ...

culture-and-maintenance-of-human-embryonic-stem-cells

Research

JoVE Journal

Biology

Culture and Maintenance of Human Embryonic Stem Cells  

47.7K ビュー.  Stemgent. このプロトコルは、健康的、未分化ヒト胚性幹（ES）細胞を維持する方法を示します。

ビデオ: Culture and Maintenance of Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

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 ...

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 ...

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.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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.

テラトーマ形成により、ヒト胚性幹細胞の運命のマッピング

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...