著者らは、小児病院、パラフィルムの染色技術で最初の命令のためにエモリー大学でhNSPC文化や博士ゲイリーBassellを提供するためのオレンジカウンティ研究所で国立ヒト神経幹細胞資源の博士フィリップH.シュワルツ氏に感謝します。

1日目：ドイツのガラス製カバースリップとその種の細胞を調製する 注 ：ドイツのガラス製カバースリップは、多くの場合、培養一次神経幹細胞および神経細胞に対して好ましい。我々は、細胞接着と拡散を改善するために以下の洗浄プロトコルを使用してください。カバーの両側に液体のアクセスを許可する小さなラック内の位置は、カバースリップ。最初に、脱イオン水で3回洗浄に続いて10分間1％Liquinoxにカバースリップを、インキュベートする。は石鹸の泡が残っていないことを確認してください。次に、30分間1M HCl中でインキュベートスリップは、脱イオン水で3回洗浄した。 65℃乾燥したカバースリップ℃で一晩。ガラス皿と滅菌するオートクレーブにカバースリップを転送します。 <ol><li>組織培養フードの中で、適切なサイズのウェルプレートに滅菌カバーグラスを置く。 </li><li>細胞接着のためのラミニンでコートカバースリップ。室温で5分間10μg/ mlのポリ滅菌水でD -リシン（PDL）でカバースリップをインキュベートする。 37℃の組織培養インキュベーターで、一晩4時間、またはのためのEMEM 20μg/ mlのラミニンでPDLとインキュベートカバースリップを取り除きます。 <ol><li> 注 ： 継代ヒト神経幹細胞の記事（への参照<a href="/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp?ID=263</a> ）とカウントヒト神経幹細胞の記事（ <a href="/index/Details.stp?ID=262">https://www.jove.com /インデックス/ Details.stp？ID = 262</a> hNSPCsを再懸濁し、カウントする方法を学ぶ）。 </li></ol></li><li>各カバースリップを含む一定の密度でも（私たちはしばしば、我々は免疫染色のためにメッキされたセルのための100,000-300,000細胞/ mlの初期播種密度を使用）にPBSとシード細胞とラミニンでコーティングされたカバースリップをすすぐ。 </li><li>場所は37のプレート℃の組織培養インキュベーターおよび細胞はカバースリップ（通常4時間の最小値が必要とされる）に付着することができます。 </li></ol> 2日目：固定、透過性、ブロッキング、および細胞に一次抗体を追加する 注 ：我々は、細胞の形態を維持するために、以下の4％パラホルムアルデヒド固定液を使用する。レシピは、パラホルムアルデヒドが溶液に入るようにするpHの移行が含まれています。より高い温度で蛍光を使用するときにバックグラウンド染色を増加させることができますギ酸の生成につながる可能性があるので、この方法ではなく、パラホルムアルデヒドを溶解するために熱の使用を好む。他の全体的な形態（ショ糖）を維持するのに役立つしながら固定ヘルプの一部のコンポーネントは、細胞骨格構造（MgCl 2及びEGTAを）維持する。 細胞を4％パラホルムアルデヒド固定液<table style="border-collapse: collapse; border: medium none" border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 試薬と手順： </td><td> 固定液の100mLのための金額： </td></tr><tr><td> MilliQ水</td><td> 75ミリリットル</td></tr><tr><td>パラホルムアルデヒド（ドラフト内で秤量） </td><td> 4グラム</td></tr><tr><td> 10N NaOHは、かき混ぜると解決策が解消されるまで滴下して加える</td><td>必要に応じて</td></tr><tr><td> 10 × PBS </td><td> 10ミリリットル</td></tr><tr><td> 1M MgCl 2の </td><td> 0.5ミリリットル</td></tr><tr><td> 0.5 M EGTA </td><td> 2ミリリットル</td></tr><tr><td>スクロース</td><td> 4グラム</td></tr><tr><td> 6N塩酸、pH7.4に滴定する</td><td>必要に応じて</td></tr><tr><td> MilliQ水</td><td> 100mlに持参</td></tr><tr><td colspan="2">最大1週間まで4℃で保存する。 37℃くらいまで温め、使用前に</td></tr></tbody></table><ol><li>ベンチで、メディアを取り出して、温めておいた固定液を加える。 5-15分（検出する抗原に応じて、我々は通常10分を使用）インキュベートする。この、室温では、次の手順を実行します。 </li><li>多くの機関が有害廃棄物としてホルムアルデヒドを取り扱うよう、適切なコンテナに固定液を捨てる。 PBS、5分ごとに時間で細胞を3回洗浄。細胞からソリューションを削除する際に、吸引は細胞を乾燥させないこと。また、いつものコートが、それらは非常に細胞が乾燥して残っていないしているソリューションを削除する前にセルを追加する一方で、次のソリューションを持っている。 </li><li>目的の抗原が細胞内にある場合、細胞膜は、抗体のエントリを可能にするために透過性にする必要があります。細胞を透過性に、PBSで新鮮な0.3パーセントトリトンX - 100溶液を使用してください。ピペットは、徐々にトリトンは粘性であり、そして洗剤が完全に細胞に添加する前に、PBSに溶解されていることを確認しますので。 PBS、5分ごとに時間で細胞を3回洗浄した細胞を5分、透過処理。 </li><li>細胞は、抗体の非特異的結合を防ぐためにブロッキング剤で処理する必要があります。我々は、ブロッキング剤としてウシ血清アルブミン（BSA）を使用してください。 （それは溶解に時間がかかるので、ロッカーやローテーター上で）ブロッキング溶液をPBSに5％BSAを溶解して作られてからゾルをフィルタリング0.45μmのシリンジフィルターでution。室温で1時間5％BSA / PBS溶液に細胞をブロックする。 </li></ol> 1％BSA / PBS（5％BSA / PBSから希釈して調製）で事前に決められた濃度に（興味の蛋白質のためうまくいけば、特定のである）一次抗体を希釈します。場所は、パラフィルムで覆われたガラス板上に一次抗体（25mmのカバースリップあたり30μL、18 mmのカバースリップあたり20μlの、12mmのカバースリップあたり15μl）を希釈した。細胞は下向きに、そして細胞が抗体と接触しているように抗体溶液の上に置くように反転させる、固定された細胞をカバースリップを拾う。構造全体での秒パラフィルムストリップを配置。 4℃で一晩インキュベートする。 3日目：洗濯、二次抗体のインキュベーション、核染色および取付け<ol><li>慎重に細胞が上直面しているので、トップパラフィルムストリップを取り外して、カバースリップをピックアップ、それを反転し、洗浄のために各ウェルにPBSでプレートに戻して置きます。 </li><li> PBS、5分ごとに時間で細胞を3回洗浄。 </li><li>一次抗体がウサギで行われた場合には、一次抗体を検出する二次抗体を選択して、例えば、、二次抗体はウサギIgGを認識すべきである。また、抗体（IgG、IgM抗体、等）抗体のアイソタイプが一致するように注意してください。複数の一次抗体を使用している場合、それらが異なる種またはアイソタイプ、および二次抗体に接続された別のマーカーを検出するために計画からのものであることを確認してください。たとえば、緑色の蛍光体に結合した抗マウス二次で赤蛍光体に抗ウサギ二次結合を使用することができます。一次と同じ技法を用いて、室温で暗所で2時間、1％BSAで適当な濃度に希釈した二次抗体溶液で細胞をインキュベート（ビデオには示されていないステップ）。二次はグリセリン溶液（25 mmのカバースリップあたり40μlの、18mmのカバースリップあたり30μL、12 mmのカバースリップあたり25μlの）として格納されている場合、インキュベーションに必要な二次抗体の量は、一次抗体のそれよりわずかに大きくなることがあります。 <ul><li> 注 ：あなたが二次抗体を可視化する蛍光分子を使用している場合は、試料が二次抗体が追加された後、光から保護される必要がある。暗闇の中で、またはフォイルカバープレートでインキュベートする。 </li></ul></li><li>一次抗体のために説明するように慎重にパラフィルムからカバースリップを取り除きます。細胞を2μg/ mlをヘキスト核PBSで希釈した染色（核の対比染色を優先する場合）で1分間のインキュベーションに続いて、PBSで2回、各時間5分を、洗う。ヘキスト染色した場合、古いですし、信号が弱過ぎないか、あなたは、インキュベーションの時間および/または濃度を増やすことができます。ヘキストとのインキュベーションの後、5分間PBSで1回洗浄する。 </li><li>カバースリップを顕微鏡で可視化するためのマウントメディアとスライド上にマウントする必要があります。蛍光分子は、二次抗体の可視化のために使用されるケースでは、マウントメディアは退色最小限に抑えるために薬剤が含まれている必要があります。我々は、蛍光用の取り付けメディアとしてVectashieldを使用してください。顕微鏡スライド上にVectashieldの適切なサイズのドロップ（25 mmのカバースリップあたり12μlの、18mmのカバースリップあたり6μL、12 mmのカバースリップあたり3μl）を置きます。 </li><li>慎重にカバースリップをピックアップし、塩を（PBSから）削除するには、脱イオン水で背中を洗う。キムワイプまたはペーパータオルで余分な水分をオフDAB、とは下に向けてセルを持つVectashieldのドロップに横たわっていた。角度でカバースリップを置き、トラップの気泡を避けるために徐々に下降することができます。日付と任意のサンプル情報を使用してスライドにラベルを付けます。 </li><li>カバーグラスとスライドはカバースリップの端の周りから（暗所で）過剰Vectashieldオフして吸引を乾燥することができます。必要に応じてカバースリップのエッジは今（カバーグラスを倒立顕微鏡で表示される場合に特に便利）、マニキュアで密封することができます。我々は通常、-20℃の冷凍庫に私たちのスライドを保存する。 </li></ol>

<ol>
	<li>Flanagan, L. A., Chou, J., Falet, H., Neujahr, R., Hartwig, J. H., Stossel, T. P., Filamin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamin+A,+the+Arp2/3+complex,+and+the+morphology+and+function+of+cortical+actin+filaments+in+human+melanoma+cells.">Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.</a> J. Cell Biol. 155, 511-518 (2001).</li><li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> J. Neurosci. Res. 83, 845-856 (2006).</li><li>Flanagan, L. A., Ziaeian, B., Palmer, T., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+stem+cells.">Immunocytochemical analysis of stem cells.</a> In Human Stem Cell Manual: A Laboratory Guide. , (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li></ol>

このプロトコルは、必要な抗体の量を最小限に抑え、信頼性の高い細胞の染色を与えるhNSPCsための免疫染色の手順を説明します。として説明する手順は、細胞内抗原に最適ですが、細胞表面分子を染色したり、細胞骨格の染色を高めるために変更することができます。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>German glass (Deutsche Spiegelglas) coverslips</td>
<td>Tool</td>
<td>Carolina Biological Supply Company</td>
<td>WW-63-3029</td>
<td>12,15,18, 22, 25 mm coverslips available</td>
</tr>
<tr>
<td>Liquinox phosphate-free detergent</td>
<td>Reagent</td>
<td>Sigma</td>
<td>Z273279</td>
<td>very viscous</td>
</tr>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, natural mouse</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>24 well plate</td>
<td>Tool</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP151-500</td>
<td>very viscous</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>potential carcinogen, use caution when using it in powder and solution form</td>
</tr>
<tr>
<td>Bovine Serum Albumin, IgG-free</td>
<td>Reagent</td>
<td>Jackson Immunoresearch</td>
<td>001-000-161</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>H-1399</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>viscous</td>
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immunocytochemistry: human neural stem cells

免疫細胞は、最も一般的に存在を決定するための非常に強力で、かなり簡単な方法、細胞内局在、及び目的の抗原の相対量、培養細胞での蛋白質、である。このプロトコルは、それらの染色質の高い、再現性の定性的および定量的データを取得中に研究者が一次および二次抗体を節約することを可能にする手順のわかりやすいシリーズを紹介。染色のために必要な抗体の量を節約するために役立つ、このプロトコルには2つの側面があります。一つは、細胞は組織培養プレートのウェルに配置されている小型の、円形のカバーグラス上で増殖されています。固定後、カバースリップ上の細胞は、プレートのウェルから除去することができます。抗体染色の場合は、セルとカバースリップをパラフィルム上に抗体溶液の小滴の上に反転し、乾燥を防ぐためパラフィルムの2番目の部分で覆われている。この方法を使用して、抗体溶液のだけ〜25μlの染色される各カバースリップ（またはサンプル）のために必要です。このプロトコルは、ヒト神経幹/前駆細胞（hNSPCs）の免疫染色について説明します、しかし他の多くの細胞型に使用することができます。

Watch this Scientific Journal Video about Immunocytochemistry: Human Neural Stem Cells at JoVE.com

Immunocytochemistry: Human Neural Stem Cells

免疫細胞が存在、細胞内局在性、および培養細胞における目的の抗原の相対量を決定する強力な方法です。このプロトコルは1つの抗体を節約し、自分の染色を最大限に活用することを可能にする手順のわかりやすいシリーズを紹介。

免疫細胞：ヒト神経幹細胞

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

immunocytochemistry-human-neural-stem-cells

Research

JoVE Journal

Biology

27.3K ビュー.  University of California, Irvine (UCI). 免疫細胞が存在、細胞内局在性、および培養細胞における目的の抗原の相対量を決定する強力な方法です。このプロトコルは1つの抗体を節約し、自分の染色を最大限に活用することを可能にする手順のわかりやすいシリーズを紹介。

ビデオ: Immunocytochemistry: Human Neural 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

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Amaxa Nucleofectorでヒト神経幹細胞をトランスフェクト

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

ヒト神経幹細胞を数える

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

ヒト神経幹細胞を継代

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

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 ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

マウス表皮神経堤幹細胞（EPI - NCSC）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have 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 ...

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

ヒト人工多能性幹細胞をトランスフェクトしNucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...