ジョナタン ルーカスは、フィーダー細胞培養、動物のケアのための Servei Estabulari デ ラ UAB とビデオを記録するための Domènec ・ マルティンと技術的な援助を感謝いたします。この作品は Ministerio デ エコノミア y Competitividad AGL2014-52408-R、ジャナラリター ・ デ ・支えられているカタルーニャ 2014 SGR 524。MVC は、PIF UAB フェローシップの受益者です。

動物と Autònoma · デ · バルセロナの人間研究に関する倫理委員会、部門 d の以下のセクションに記載されている動物の使用を承認されている &#39; Ramaderia、育種釣り私ジャナラリター ・ デ ・ カタルーニャ (プロトコル #8741) の Alimentació。
 1 フィーダー細胞の不活性化とストレージ 注: ひと包皮線維芽細胞 (HFF-1) 以前 90% 胎仔ウシ血清 (FBS) と 10% ジメチル ジメチルスルホキシド (成る凍結溶液 1 mL でフリーズした。DMSO)、使用するまで液体窒素に保存されます。
<ol><li>37 ° C の水浴のバイアルを浸すことによって HFF 1 (1 mL) のセラムを解凍</li>。
	<li>極低温バイアルの解凍の内容を予め温めておいたダルベッコに追加 &#39; 更新イーグル s &#39; s 媒体 (DMEM) 10% を 1:10 に FBS 希釈</li>。
	<li>T75 フラスコで希薄化後のセル (10 mL) をシードし、37 ° C、5% CO 2 でそれらを文化します</li>。
	<li>次の日は、DMSO の残党を排除するために媒体を変更します。メディアを削除し、10% に予め温めておいた DMEM の 10 mL を追加 FBS</li>。
	<li>、タイトピロー包装文化し、文化融合の段階に達するまで、媒体ごとの 48 h を変更します。これは約 7 〜 10 日かかる場合があります</li>。
	不活化ソリューションを準備、10% を DMEM にマイトマイシン C を希釈する <li>10 μ G/ml の最終的な集中に FBS。37 ° C、5% CO 2 で 3 h の不活化ソリューションと細胞をインキュベートします</li>。
	<li>不活性化ソリューションを削除、三度と 2 mL のハンクス平衡塩ソリューション (HBSS) マイトマイシン C の残骸を除去するために細胞をすすいでください</li>。
	<li>不活化セルを解除するには、予め温めておいたトリプシン EDTA 溶液の 1 mL を追加し、37 ° C、5% CO 2 で 5 分間インキュベートします</li>。
	<li>が切断されていることを確認する倒立顕微鏡下で細胞を観察 (0.3 の開口または NA 10 × 対物)。そうでない場合は、37 ° C、5% CO 2 で、数分間インキュベートします。再び倒立顕微鏡下で細胞を確認してください</li>。
	<li>細胞塊を分離するためにソリューションをパスツール ピペット ピペットします</li>。
	<li>10 %fbs と再懸濁します DMEM の 4 つの mL を追加することによって酵素のソリューションを中和します</li>。
	<li>細胞懸濁液の因数と HBSS で 1: 100 希釈を行います。セルの数を決定する、ノイバウアーに希釈の 10 μ L を読み込む &#39; s 商工会議所。(0.45 NA で 20 × 対物) 倒立顕微鏡下で商工会議所の大きな四角形の 1 つであるセルをカウントします。
	<ol><li>4 つの大きな正方形の合計にカウントの繰り返し。N (N) のセルの合計数を計算するセルの数の平均値を = 容量希釈 x 10 4 x x カウント</li>。
	</ol></li> <li>上清 200 x g. 破棄で 5 分間サンプルの残りの部分を遠心し、凍結 1 mL あたり 10 の 6 セルの濃度の溶液にペレットを再懸濁します</li>。
	<li>因数 10 6 セル (1 mL) 各セラム チューブの懸濁液。冷凍コンテナーでセラム チューブを置き、-80 でフリーズ ° C</li>
	<li>次の日は、液体窒素でセラム チューブを格納します</li>。
</ol> 2。フィーダー細胞培養 <ol><li>導出プロセスを開始する前に 1 〜 4 日雪解けセラム 1 x 10 の 6 を含む不活化 37 で水浴の HFF 1 セル ° C</li>
	<li>で解凍 HFF 1 の 1:6 希釈加温 10 %fbs と 37 温 DMEM を ° C</li>
	<li>豚皮から予め温めておいた 0.2% のゼラチンの 500 μ L で 4 ウェル プレートのすべての井戸を埋めるし 37 ° C で 10 分間でプレート</li>
	<li>は井戸からゼラチンの 500 μ L を取り外してタイトピロー包装希釈を 500 μ l 添加します。井戸の底にタイトピロー包装の均一な分布を確実にプレートがみ合います。円形の動きを避けるために、井戸の中央に細胞が集中</li>。
	<li>少なくとも 24 h 各ウェルで不活化タイトピロー包装の単分子膜を取得する CO 2 を 37 ° C、5% でプレートをインキュベートします</li>。
	<li>次の日は (0.3 NA と 10 × 対物) 倒立顕微鏡下で細胞を観察し、接続されているが、均一を確認します。DMSO の残党を排除するために媒体を変更します。メディアを取り出して、10% に予め温めておいた DMEM を 500 μ l 添加 FBS</li>。
</ol> 3。胚のコレクションおよび文化 <ol><li>4 日採卵前に、管理 5 IU 妊馬の &#39; 6-12 週齢の雌マウスに腹腔内投与による s 血清ゴナドトロピン 。
	注: 他のマウス系統から女性を使用できますが、129S2 または B6CFAF1 株から雌を使用している我々。それにもかかわらず、とき (例えば CBA)、非寛容な株から雌と作動媒体補わなければなりません 2i と手順 4.2 で説明した</li>。
	<li>後 48 h と腹腔内投与によるひと絨毛性ゴナドトロピンの 5 IU を管理する少なくとも 8 週齢、1:1 の比率で男性と女性を仲間します。次の日、男性から女性を区切ります</li>。
	<li>採卵前日は 35 mm 細胞培養シャーレ 4 mg/ml のウシ血清アルブミン (BSA) KSOMaag 培のいくつかの 40 μ L 滴を準備し、鉱物油滴を完全にカバーするためにプレートを埋めます。媒体を平衡に CO 2 を 37 ° C、5% で皿をインキュベートします</li>。
	胚操作ピペットを準備する <li>は熱長い先端ガラス パスツール ピペット ブンゼン バーナーを使用しての薄い部分です。柔らかい、炎から引き出すし、2 つの端を離れて引っ張る。希望の長さにそれを破る。先端の内径が 100-150 μ m にする必要があります</li>。
	<li>頚部転位 45 48 h の ポストの coitum (パソコン) による雌マウスを安楽死させる</li>。
	<li>は、湾曲した滑らかな鋭いハサミで皮膚とまっすぐ鋭いハサミで腹膜を切断することによって腹腔内を公開するマウスを解剖します。ピンセットで 1 つ子宮をつかむ、まっすぐ鋭いはさみを使用して卵管を切断し、Hepes バッファー CZB 培地 (HCZB) 13 37 に ° c. その他の卵管を削除する処理を繰り返します</li>。
	2 細胞期胚を収集する時計鉗子によって行われて <li>フラッシュ HCZB 搭載 1 mL 注射器で卵管 。
	注: フラッシュの針を 30 G の注射針を準備する、最後をカットし、研磨石の鈍い先端に地面します</li>。
	<li>口のピペットなどの器具を使用して、胚をピックアップ、HCZB で洗って、胚盤胞期まで 37 ° C、5% CO 2 で事前に準備された胚培養皿にそれらを文化します。50 胚までそれぞれ 40 μ L ドロップで培養することができます。胚開発顕微鏡の下のすべての 24 h を監視します 。
	注: 代わりに、胚を 体外で 胚培養を短縮する胚盤胞の段階で収集できます。この場合、3.5 から 4.5 p. c. の日に女性を安楽死させる、子宮を解剖します。HCZB とフラッシュと</li>。
</ol> 4。幹細胞誘導 <ol>胚の播種日 派生プレートの準備 <ol><li><li>100 μ M β 2 メルカプトエタノール、1 を DMEM 培地を補うことで派生メディアの準備 x 非必須アミノ酸、血清の置換、および 10 の 20 3 U/mL LIF。</li>
		<li>に応じて、また追加 MEK 阻害剤 PD0325901 の 1 μ M と 3 μ M の GSK3 阻害剤 2i 媒体を取得する CHIR99021 です</li>。
		<li>取るから不活化タイトピロー包装 (ステップ 2.6) の単分子膜 4 ウェル プレート、インキュベーター。メディアを取り出して、予め温めておいた派生中の 800 μ L を各ウェルに追加します。インキュベーターにプレートを返します</li>。
	</ol></li> <li>透明帯除去、播種胚 <ol><li>35 mm 細胞培養シャーレ酸性 Tyrode 3 30 μ L 滴と準備 &#39; s ソリューション 14 派生中 3 30 μ L 滴。鉱物油と滴をカバーし、37 で皿を保つ ° C</li>
		<li>は、インキュベーターから胚培養皿を取る。一つずつ酸性 Tyrode 文化滴から、胚盤胞を転送 &#39; s ソリューション ドロップ操作、胚のピペットを使用しています。可能な場合のみ、拡張胚盤胞導出プロセスを開始するを選択します</li>。
		<li>は、顕微鏡下で透明帯消化を監視します。透明帯が消えると、すぐに、派生中ドロップする酸性 Tyrode を洗浄するために胚盤胞を転送 &#39; s ソリューションです</li>。
		<li>各ウェルにインキュベーターと種子 1 つ透明帯除去胚盤胞フィーダーから 4 も派生板細胞を取る</li>。
		<li>4 よく導出プレート インキュベーターと 37 ° C、5% CO 2 で文化に戻ります</li>。
	</ol></li> <li>監視幹細胞成長および媒体を変更 <ol><li>培養倒立顕微鏡 (0.45 NA で 20 × 対物) の下のすべての 24 h を監視します。幹細胞の分化、腫れの屈折細胞周囲の副産物かもさておき、フィーダー細胞を押すなどの兆候があるかどうかに注意することが重要だ</li>。
		<li>毎日、文化の媒体を変更します。インキュベーターから 4 ウェル プレートを取る、各ウェルからメディアを取り出して、使用される場合は LIF と 2 i、予め温めておいた導出培の 800 μ L を追加します。(6-8 日) 観察される増殖までの文化を維持します</li>。
	</ol></li></ol> 5。幹細胞文化およびメンテナンス <ol><li>準備 Kolle のようなハンドルおよび幹細胞操作ピペット。フォロー ステップ 250-300 μ m の先端の内径と幹細胞操作ピペットを準備するのに 3.4 Kolle のようなハンドルを準備するのにプルのパスツール ピペットの他の部分を使用します。熱し、フックを得るまで形状します</li>。
	<li>は、インキュベーター外 4 ウェル培養プレートを取るし、顕微鏡下で伸長を探します。分化細胞や副産物を囲む餌箱を押してすぐハンドルを使用します 。
	メモ: 最終的な分化、伸長を避けるためには、それは副産物の端から差別化されたすべてのセルを削除することが重要です</li>。
	<li>幹細胞操作ピペットと伸長を吸引し、50 μ L のトリプシン-EDTA ドロップで新しい料理</li>。
	<li>メスを使用して、両方の機械的および酵素の分解およびそれのためにいくつかの部分に突起をカットします</li>。
	<li>すぐにトリプシンを中和するために導出中の新鮮なドロップに伸長フラグメントを転送します</li>。
	<li>は、フィーダーの単分子膜の新しい 4 ウェル プレートに伸長フラグメントを転送します。ウェルあたり最大 10 個の断片を転送し、誘導培地で 37 ° C および 5% の CO 2 とそれらを文化します</li>。
	<li>は、mESC の樹立を検討する前に、少なくとも 6 週間の幹細胞のような文化を維持します。他のすべての日とサブカルチャー媒体を変更して週に 1 回</li>。
</ol>

<ol>
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M., et al. <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+culture+conditions+support+distinct+states+associated+with+different+developmental+stages+and+potency.">Embryonic stem cell culture conditions support distinct states associated with different developmental stages and potency.</a> Stem Cell Rep. 7 (2), 177-191 (2016).</li></ol>

著者が明らかに何もありません。

MESC 行派生は日常的に多くの実験室で使用されるよく知られているプロシージャが、その効率は常に多能性維持を妨げることができる複数の要因のために期待高。本稿で示す、ステップバイ ステップ、シンプルで信頼性のあるプロトコルを使用して高効率 mESC ラインを確立する方法。これらの効率は文献で報告されたものに似ています。
最大の効率を確保するため、毎?...

胚性幹細胞 (ESC) は、自己更新または [特定の条件1を区別する能力を保持する着床前胚由来多能性細胞です。これらのプロパティに基づいて、esc キー、再生医療、疾患モデル作製、組織工学研究2便利なツールとなっています。
ESC は、初めて着床前マウス胚、低成功3,4マウス ESC (mESC) 線を元から派生しました。長年、導出効率残った多能性体外を維持が困難なのため、低。その上フィーダー細胞の5導出の効率を向上させる存在促進コロニーの添付ファイルと、核型安定性6、多能性維持の改善にプロトコル リードのいくつかの変更。最も重要なだった白血病抑制因子 (LIF) のまた mESC 培7、8、129S2 寛容な背景9からの胚の使用と mESC を添加した培地での培養が定義されています。血清、潜在的な差別化要因10の無料。最近では、説得力のある証拠はまた mESC 培 (2 i として知られている) するシグナル伝達経路の 2 つの小分子変調のカクテルの多能性を維持するために最善であることを示しています。マイトジェン活性化タンパク質キナーゼ (MAPK) 経路を阻害することによって分化信号を減らす、PD0325901 MEK 阻害剤の GSK3β 阻害剤 CHIR99021 は、低密度で細胞の生存を高める組み合わせから成るカクテル 2i11wnt シグナル経路を活性化。
本稿では効率的に mESC 行をマウス胚盤胞に由来する単純なプロトコルをについて説明します。LIF と血清交換12導出培にフィーダー細胞に寛容な系統からの胚を培養標準の手順を従ってください。このプロトコル、mESC ラインは、2 i の中で得られたものと同等の効率で派生できます。これにもかかわらず、非寛容な系統を扱うときに多能性維持、または可能性のある問題を回避する 2 i を追加できます。

<table border="0" cellpadding="0" cellspacing="0" width="1125">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">1 mL sryringe</td>
			<td style="width:199px;">BD&#160;</td>
			<td style="width:141px;">309628</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">2-&#946;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">4-well plate</td>
			<td>Thermo Scientific Nunc</td>
			<td style="width:141px;">176740</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:263px;">Acidic Tyrode&#39;s solution&#160;</td>
			<td style="width:199px;">Home-made</td>
			<td style="width:141px;"></td>
			<td style="width:524px;">See recipe in Nagy et al, 2003. For short-term storage (up to 1 month) keep it at 4&#186;C. Alternatively, for long-term storage keep it at -20&#186;C. If the efficiency of the solution diminishes, it should be acidified by the adition of a small drop of HCl 1M.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">BSA</td>
			<td>Sigma</td>
			<td style="width:141px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:263px;">Chicken anti-Mouse IgG, Alexa Fluor 488</td>
			<td>Molecular Probes - Invitrogen</td>
			<td style="width:141px;">A-21200</td>
			<td>1:500 dilution. Secondary antibody for Oct4, Tuj1, &#945;SMA and AFP mouse antibodies.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:263px;">CHIR990212</td>
			<td style="width:199px;">Axon Medchem&#160;</td>
			<td style="width:141px;">1386</td>
			<td style="width:524px;">Reconstitute with 2.15 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20&#186;C. Avoid freeze and thaw cycles.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">CryoTube</td>
			<td>Thermo Scientific Nunc</td>
			<td style="width:141px;">3754518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMSO</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:141px;">41640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMEM</td>
			<td style="width:199px;">BioWest</td>
			<td>L0107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">FBS</td>
			<td style="width:199px;">BioWest</td>
			<td>S1810</td>
			<td>Inactivate it prior to use by heating for 30 mins at 56&#186;C. Aliquot and store at -20&#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Flushing needle</td>
			<td style="width:199px;">BD&#160;</td>
			<td>304000</td>
			<td>Cut the end of the needle and ground to a blunt tip on an abrasive stone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Gelatin from porcine skin</td>
			<td style="width:199px;">Fluka</td>
			<td>48724</td>
			<td>Dilute at 0,2% in destilled water and autoclave it. Each dilution can be used for a month.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Goat anti-Rabbit IgG, Alexa Fluor 594</td>
			<td>Molecular Probes - Invitrogen</td>
			<td style="width:141px;">A-11037</td>
			<td>1:500 dilution. Secondary antibody for Sox2 rabbit antibody.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">HBSS</td>
			<td style="width:199px;">BioWest</td>
			<td style="width:141px;">L0611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Hepes-buffered CZB&#160;</td>
			<td style="width:199px;">Home-made</td>
			<td style="width:141px;"></td>
			<td>See composition in Chatot et al, 1989</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Human chorionic gonadotropin</td>
			<td style="width:199px;">Divasa-Farmavic</td>
			<td style="width:141px;">Veterin-Corion 3000 UI</td>
			<td style="width:524px;">Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&#186;C for up to two months. Once thawed do not freeze again.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human foreskin fibroblasts</td>
			<td style="width:199px;">ATCC</td>
			<td style="width:141px;">ATCCSCRC-1041</td>
			<td>For long term storage it is recommended to store them in liquid nitrogen.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:263px;">KnockOut Serum Replacement</td>
			<td style="width:199px;">Life Technologies</td>
			<td>&#160;10828-028</td>
			<td style="width:524px;">Photosensitive. It is recommended to aliquot it in small volumes such as 10 ml and store it at -20&#186;C (check the expiration date). Avoid freeze and thaw cycles.&#160;&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">KSOMaag Evolve</td>
			<td style="width:199px;">Zenith Biotech</td>
			<td style="width:141px;">ZEKS-050</td>
			<td style="width:524px;">Supplement with 4 mg/ml BSA. Equilibrate at 37&#186;C and 5% CO2 a day prior to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Leukemia Inhibitory Factor</td>
			<td style="width:199px;">Merk Millipore</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:263px;">Mice&#160;</td>
			<td style="width:199px;">Charles River/Harlan</td>
			<td style="width:141px;"></td>
			<td style="width:524px;">It is recommended to use mice from a permissive strain in terms of mESC derivation (such as 129S2 or C57BL). However, inbreed strains are less efficient producing embryos. Therefore, it is recomended to use a hybrid strain, such as 129S2 x C57BL or B6CBAF1 to take advantage of the hybrid vigor.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mineral oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td>Embryo tested. Photosensitive.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:263px;">Mitomycin C</td>
			<td style="width:199px;">Serva</td>
			<td>2980501</td>
			<td style="width:524px;">Reconstitute the powder with MilliQ water at 0.5 mg/ml. Store at 4&#186;C protected from light. Attention, it is harmful and suspected of causing cancer.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse anti-tubulin &#946; III (Tuj1)</td>
			<td>Biolegend</td>
			<td style="width:141px;">MMS-435P</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse monoclonal anti-&#945;SMA</td>
			<td>Sigma</td>
			<td style="width:141px;">A5228</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse monoclonal anti-AFP</td>
			<td>R&#38;D Systems</td>
			<td style="width:141px;">MAB1368</td>
			<td>1:50 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse monoclonal anti Oct3/4&#160;</td>
			<td>Santa cruz</td>
			<td style="width:141px;">Sc-5279</td>
			<td>1:50 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Non-Essential Amino Acids</td>
			<td>Life Technologies</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:263px;">PD0325901</td>
			<td style="width:199px;">Axon Medchem&#160;</td>
			<td style="width:141px;">1408</td>
			<td style="width:524px;">Reconstitute with 2.08 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20&#186;C. Avoid freeze and thaw cycles.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Petri dish (35mm)</td>
			<td>Thermo Scientific Nunc</td>
			<td style="width:141px;">153066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Petri dish (60mm)</td>
			<td>Thermo Scientific Nunc</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Pregnant mare&#39;s serum gonadotropin</td>
			<td style="width:199px;">Foligon</td>
			<td>Foligon-1000 UI</td>
			<td style="width:524px;">Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&#186;C for up to two months. Once thawed do not freeze again.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Rabbit policlonal anti-Sox2</td>
			<td>Merck Millipore</td>
			<td style="width:141px;">AB5603</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">T75 falcon</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:141px;">130190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Trypsin-EDTA 10x</td>
			<td style="width:199px;">BioWest</td>
			<td style="width:141px;">X0930</td>
			<td>Dilute 1:10 in HBSS to obtain a 1x solution</td>
		</tr>
	</tbody>
</table>

derivation of stem cell lines from mouse preimplantation embryos

マウス胚性幹細胞 (mESC) の導出は、着床前の胚からの多能性細胞が確立されたプロセスです。これらの行は、自己更新または特定の条件の下で区別するための能力を保持します。これらのプロパティにより mESC、再生医療、疾患モデル作製、および組織エンジニア リングの研究に有用なツールです。寛容なマウス系統白血病阻害因子を添加した培地でフィーダー細胞から胚盤胞培養 mESC 行高導出効率 (60 ~ 80%) を取得するための単純なプロトコルを説明します。プロトコルは、派生メディア (2i 中) に 2 つの小分子阻害剤のカクテルの簡単な付加によって効率的に mESC 行を非感受性マウス系統から派生にも適用できます。準備とフィーダー細胞、コレクションと mESC 行のマウス胚および派生のカルチャとカルチャのカルチャに関する詳細な手順を提供しています。このプロトコルは、特殊な機器を必要としないし、基本的な哺乳類の細胞の文化の専門知識を持つ任意の研究室で実施することができます。

このプロトコルに従い以上伸長形成の 95% の文化 (図 1 a) の最初の週後達成する必要があります。6 継代後 mESC の樹立は、60-80% の平均成功の実験的複製間で変数です。2i 添加ほど向上しません結果 129S2 のバック グラウンドを持つものなど、寛容なマウス系統を操作するときが、非寛容な系統を使用するときに必要です。
<p class="jove_content" fo:ke...

Watch this Scientific Journal Video about Derivation of Stem Cell Lines from Mouse Preimplantation Embryos at JoVE.com

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

マウス着床前胚幹細胞の誘導

この資料では、効率的に派生し、胚盤胞の段階でマウス胚からの多能性幹細胞株を培養するプロトコルについて説明します。

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

derivation-of-stem-cell-lines-from-mouse-preimplantation-embryos

Research

JoVE Journal

Developmental Biology

8.3K ビュー.  Universitat Autònoma de Barcelona. この資料では、効率的に派生し、胚盤胞の段階でマウス胚からの多能性幹細胞株を培養するプロトコルについて説明します。

ビデオ: Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

胚性幹細胞から心臓前駆細胞の誘導

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

発生生物学

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

幹細胞から網膜色素上皮細胞の効率的な導出

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

小分子変調分化とその後のグルコース飢餓を用いたヒト人工多能性幹細胞からの高度に精製された心筋細胞の導出

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

ゲノム編集したヒト多能性幹細胞株の樹立：分離へのターゲティングから

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

マウス着床前胚でリネージュの仕様を研究するためのタンパク質発現の定量分析

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

loss-が及び着床前マウス胚の初期の細胞運命決定要因を調査するための機能獲得型のアプローチ

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

ヒト多能性幹細胞からメラノサイトのフィーダーフリーの導出

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

ゼブラフィッシュ胚からの内因性受容体の親和性ラベリング検出

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

トランスポゾンのシステムを用いたマウスの羊水細胞から多能性幹細胞の作製

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

ラミニン521マトリックスを用いたヒト誘導多能性幹細胞の統合された誘導

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...