1。解凍hES細胞プレ実験のセットアップ<ol><li>一日前に解凍hES細胞に、プレートMEF培地でゼラチンコート6ウエル組織培養プレート（D - MEM基礎培地を添加した1つのウェル上に照射したマウス胚性線維芽細胞（MEF）、CF1系統、のフィーダー層10％熱不活性化FBS、1％非必須アミノ酸）。 37℃および5％CO 2を一晩インキュベートする。 </li><li>液体窒素からヒトES細胞を除去する前に、場所と効率的かつ成功した融解を確保するために使用する準備に必要なすべての機器と試薬を用意してください。 </li></ol> hES細胞を融解<ol start="3"><li>金属製のピンセットを用いて液体窒素貯蔵タンクからのヒトES細胞の極低温バイアルを取り外します。 </li><li>霜を取り除くために3〜5秒のために手袋をはめた手の間にバイアルを転がし。 </li><li>バイアルのラベルに情報を記録します。 </li><li>金属製のピンセットを使用して、37℃の水浴中にバイアルを浸す。スワール優しくバイアルおよび氷結晶のサイズを確認するには、光にバイアルを保持することによって（ただしすぐに！）多くの場合、解凍の進行状況を観察。沈める水浴中でバイアルのキャップを、これは細胞を汚染する可能性として使用しないでください。 </li><li>唯一の小さな氷の結晶が残っている、水没殺菌にエタノールで全体バイアル。 </li><li>無菌生物学的安全キャビネット内で、15 mLコニカルチューブの底に直接低温バイアルの内容を転送します。 </li><li>徐々にヒトES細胞培養培地を4mL（20％ノックアウト血清代替物、1％非必須アミノ酸、1％L -グルタミン、0.2％β-メルカプトエタノール、4 ngの/を補充D-MEM/F-12基礎培地を追加するチューブへの添加のbFGF）。ゆっくりと新しいメディアがチューブに追加されるよう継続的に細胞を混合して揺らし。 </li><li> 200 × gで5分間、細胞を遠心分離</li></ol> hES細胞をめっき<ol start="11"><li>細胞株、継代数、および融解日付：ヒトES細胞は遠心分離している一方で、適切​​なhES細胞の情報で標識することにより、MEFフィーダープレートを準備。 </li><li>スピン時に残された約2分がある場合には、MEFの培地を吸引し、ウェルにPBS 1.0 mLを加える。 </li><li>生物学的安全キャビネットに戻ってペレット化hES細胞を持参し、慎重に上清を吸引除去する。細胞ペレットを吸引しますが、このソリューションは凍結培地からDMSOが含まれているように、上清をできるだけ削除しないように注意してください。 </li><li>ペレットは、5 mLのガラスピペットを用いてヒトES細胞培養の培地2.5 mLを加えると3-4回ピペッティングして、非常に穏やかに再懸濁する。 </li><li>よくMEFフィーダーからPBSを吸引除去し、徐々に6ウェルプレートのウェル準備するためにヒトES細胞懸濁液のすべての2.5 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。中央のコロニーを集中避けるために、円を描くように旋回プレートをしないでください。 </li><li>細胞は37℃、一晩5％CO 2を添付することができます。 </li><li>雪解け後の日、培地を除去（あらゆる浮遊細胞を使用）と同様に新鮮なhES細胞の培養液の2.5 mlを加える。 </li><li>オプション：削除された培地と細胞が別々の準備MEFフィーダープレートに転送することができます。このステップでは、多くの場合、元の融解から細胞と並行して培養することができる新たなコロニーを生成します。 </li><li> 37℃、5％CO 2で一晩培養する。 </li></ol> 2。 hES細胞の凍結<ol><li>ヒトES細胞は遠心分離されている間、細胞株、継代数、および凍結日付を含む生物学的安全キャビネット内部の極低温バイアルを、ラベルを付けます。凍結ヒトES細胞の典型的な密度は、極低温バイアル当たりの細胞の1つのウェル（6ウェルプレートから）です。 </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、チューブから上清を吸引除去する。 </li><li>極低温バイアルあたり0.5mLのヒトES細胞培養用培地：ヒトES細胞培養培地の適切な量で細胞ペレットを再懸濁します。 hES細胞は、比較的大きなコロニーの部分で凍結時より回復として、ピペッティング時に非常に穏やかである。 </li><li>ゆっくり、静かにチューブを振とうしながら、細胞に2XヒトES細胞凍結培地の適切な量（クライオバイアルあたり0.5mL）を（FBS、20％ヒトES細胞培養培地、および20％DMSO定義されている60％）を加える。一度凍結培地をピペットで混合するために1〜2回は非常に優しく、ソリューションに追加されます。 </li><li>迅速かつ慎重に、各極低温バイアルに細胞懸濁液1 mLを加える。 </li><li>のイソプロパノール冷凍コンテナと所定の位置にバイアルを移す-80℃冷凍庫で一晩。細胞は、1 ° Cイソプロパノール冷凍コンテナで毎分フリーズします。 </li><li>翌日、すぐに金属製のピンセットを用いて液体窒素貯蔵タンクに凍結バイアルを移す。 </li></ol>代表的な結果ヒトES細胞を融解している最初の日の後、小さなコロニーが透明に表示されることがありますし、顕微鏡下で確認することが困難な場合があります。新たに融解したES細胞はゆっくり増殖する傾向があるので、彼らは確立されたコロニー（図1）として表示されるまで数日かかることがあります。 図1。液体窒素から解凍した細胞の回復と成長。hES細胞は、撮像した 1、解凍後7および11日間。

<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><li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <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), 1145-1147 (1998).</li></ol>

付属のビデオは、hES細胞を解凍して凍結する方法を示しています。液体窒素で凍結した細胞は、最適な回復を得ることが可能な限り迅速にお風呂を解凍してください。融解細胞をピペッティング時に非常に慎重に覚えている：細胞のハンドリングを最小限に抑え、穏やかにピペッティング。 hES細胞の1バイアルを6ウェルプレート、または4ウェルプレートの全ウェルのもどちらか一方に播種し、直ちにインキュベーター内に配置することができます。 4つの別々のウェル中の培養は汚染全体の文化を失う可能性を減らし、そしてより簡単に、面積比でhES細胞のコロニーを見つけるために作られることになるので、初めてのためにヒトES細胞を解凍するとき、4ウェルプレートをお勧めします。 hES細胞を融解後の最初の日、コロニーは小さく、透明であってもよい。それが確立されたコロニーとして出現するヒトES細胞の培養に数日かかる場合がありますので、コロニーは、顕微鏡下で明らかにされていない場合でも、毎日hES細胞の培養液を補充。コロニーは、多くの場合12日間解凍後10日まで継代のための適切なサイズに達していないとして、それは、細胞を解凍するときに新鮮なメッキMEFフィーダー層を使用することが重要です。 hES細胞の低継代バイアルを解凍するとき、それは将来の文化や実験のための低継代細胞のストックを維持するために追加のバイアルを拡大し、凍結することをお勧めします。また、日常的に凍結ストックを維持するためにすべての&quot;余分な&quot;健康なヒトES細胞を凍結することをお勧めします。最も成功して融解し、その後の培養は、積極的にhES細胞のコロニーを分割し、未分化、高品質として凍結した。大きな細胞凝集体として緩やかに処理した場合はhES細胞は、より効率的に凍結から回復。最終的な集合体のサイズは、ルーチンの経過後に播種凝集体のサイズ（またはより少し大きい）のようになります。

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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 mediumjavascript:void(0);</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 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>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2560</td>
<td>For freezing medium</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 pipettes</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>Cryogenic vials</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>5000-1020</td>
<td>1.5 mL capacity</td>
</tr>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5100-0001</td>
<td>&#x201C;Mr. Frosty&#x201D;</td>
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freezing and thawing human embryonic stem cells

ジェームズトムソン以来<em&gt;ら</em&gt;文化の中でヒトESを分離し、成長し、1998年の技術を開発し、後で使用すると凍結ストックからの細胞を解凍し、拡張するための凍結細胞は、ルーチンヒトES細胞培養で行われる重要な手続きとなっている。ヒトES細胞は凍結融解のストレスに対して非常に敏感であるため、特別な注意が必要です。ここでは、急速に、液体窒素の在庫からヒトES細胞を融解マウス胚性フィーダー細胞上にめっき、ゆっくりと長期保存のためにそれらを凍結するための適切な手法を示す。

Watch this Scientific Journal Video about Freezing and Thawing Human Embryonic Stem Cells at JoVE.com

Freezing and Thawing Human Embryonic Stem Cells

ジェームズトムソンらが文化の中でヒトESを分離し、成長し、1998年の技術を開発して以来、後で使用するためと凍結ストックからの細胞を解凍し、拡大して凍結細胞は、ルーチンヒトES細胞培養で行われる重要な手続きとなっている。ヒトES細胞は凍結融解のストレスに対して非常に敏感であるため、特別な注意が必要です。ここでは、急速に、液体窒素の在庫からヒトES細胞を融解マウス胚性フィーダー細胞上にめっき、ゆっくりと長期保存のためにそれらを凍結するための適切な手法を示す。

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 ...

freezing-and-thawing-human-embryonic-stem-cells

Research

JoVE Journal

Biology

45.7K ビュー.  Stemgent. ジェームズトムソンらが文化の中でヒトESを分離し、成長し、1998年の技術を開発して以来、後で使用するためと凍結ストックからの細胞を解凍し、拡大して凍結細胞は、ルーチンヒトES細胞培養で行われる重要な手続きとなっている。ヒトES細胞は凍結融解のストレスに対して非常に敏感であるため、特別な注意が必要です。ここでは、急速に、液体窒素?...

ビデオ: Freezing and Thawing Human Embryonic Stem Cells

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

凍結ヒトES細胞

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

生物学

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

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 ...

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

トランスポートのための凍結、融解、およびパッケージング細胞

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

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 ...

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 ...

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 ...

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 ...