著者は、ご提供する試薬およびマウスの博士 Minghan トンと加藤をありがとうございます。私たちは有益な議論のゾウの研究室のメンバーに感謝します。この作品は一部の助成金によって支えられた 973 プログラム中国の省の科学と技術 (ほとんど) [2014CB964704、2015CB964503] からの第 9 人民病院、上海 Jiao はさみ大学医学部の臨床研究プログラム。細胞生物学および生物化学の中核施設、上海生物化学および細胞生物学、中国科学アカデミー研究所分子細胞科学の卓越性のための CAS センターの中核施設の助けをありがとう。

動物に関する各種手続きスタンフォード紛争処理パネル研究所動物ケア (APLAC) のによって承認されたプロトコルに従って行われ、動物愛護、上海研究所の生化学と細胞の利用委員会によって承認されました。生物学。
1. 準備
<ol><li>破骨細胞特定ラプター削除マウス (ラプターフロリダ州/フロリダ; を生成します。Ctsk cre、 Ctskラップ来世)ラプターフロリダ州/フロリダ州 Ctsk creマウス マウスの交配による。本研究では WT コントロールとしてラプターフロリダ州/フロリダマウスを使用します。</li>
	<li>分離骨を保つために氷が付いている容器があります。</li>
	<li>
培養液を準備します。
	<ol>
<li>1 x グルタミン、ペニシリン ・ ストレプトマイシン, 10% 牛胎児血清と最小必須培地アルファ (α-MEM) を補うことで α MEM 培地を準備します。</li>
		<li>骨髄マクロファージ誘導培地 20 ng/mL で α MEM 培地および M CSF の 50 mL の構成を準備します。</li>
		<li>破骨細胞誘導培地から成る M-CSF と RANKL 20 ng/ml、それぞれ α MEM 培地 50 ml を準備します。</li>
	</ol>
</li>
	<li>
トラップに汚れる前に次のバッファーを準備します。
	<ol>
<li>0.8 mL の 37% (巻/巻) ホルムアルデヒド溶液クエン酸溶液 2.5 mL、アセトンの 6.5 mL から成る修正ソリューションを準備します。</li>
		<li>トラップの染色液を準備します。<ol>
<li>37 ° c. に脱イオン水を prewarm します。</li>
			<li>1.5 mL マイクロ チューブに高速ガーネット GBC 基本ソリューションの 100 μ L との亜硝酸ナトリウム溶液 100 μ L を加え、混ぜる 30 s. 休暇の穏やかな逆解析による 2 分放置する混合物。</li>
			<li>脱イオン水 37 ° C の prewarm 9 mL1.4.2.2、ナフトール AS BI リン酸液、酢酸溶液を 400 μ l 添加、酒石酸溶液 200 μ L を 100 μ l 添加のステップから高速ガーネット ジアゾ GBC 基本ソリューションの 200 μ L を追加します。</li>
			<li>37 ° C の水浴中で混合液を染色トラップを維持します。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
培養液中のトラップ活動定量化する前に次のバッファーを準備します。
	<ol>
<li>酒石酸バッファー (50 mL) を準備: 0.33 M L - 酒石酸、0.33 M 酒石酸二塩基ナトリウム 48 mL の 2 mL の水分を取り除きます。4.9 に pH 値を調整します。</li>
		<li>4-ニトロフェニル リン酸二ナトリウム (同時) バッファー (200 mL) を準備: 180 mL の脱イオン水、ZnCl2、27.2 mg MgCl241 mg グリシンの 1.502 g。3 M NaOH で 10.4 に pH 値および脱イオン水と 200 mL にボリュームを調整します。</li>
		<li>(1 つの 96 ウェル プレート) の脱燐酸化酵素の基質との同時バッファーを準備: ホスファターゼ基板のステップ 1.5.2 の同時バッファーの 175 μ L 49.37 mg。</li>
		<li>基板バッファー (9 mL/96-ウェル プレート) を準備: 6.24 mL の脱イオン水, 酢酸溶液の 360 μ L と 2.4 mL の酒石酸バッファー。渦によって混合し、使用前に 37 ° C で予熱します。</li>
		<li>予熱した基板 buffer(1.5.4) 基板の混合物と 10 の渦の 9 ml に, 耐バッファーとホスファターゼ substrate(1.5.3) の 90 μ L を追加 s。</li>
	</ol>
</li>
</ol>2. 郭清 (日-1)
<ol><li>CO2 3 1 ヶ月齢の雌の WT とラップCtskマウスを別々 に安楽死させます。訓練された人員によって適切な装置を持つ CO2吸入を実行します。</li>
	<li>75% エタノール (巻/巻) 細菌汚染を防止し、仰臥位で郭清ボードの上に 5 分間の 100 mL のビーカーに 6 匹のマウスを浸します。脛骨の遠位部に縦切開をするそして眼科用ハサミで後肢に沿って皮膚を解剖します。</li>
	<li>股関節の関節の靭帯をハサミで切って、トランクから後肢を脱臼します。</li>
	<li>膝関節の関節の靭帯を切り、慎重に脛骨と大腿骨、膝関節から関連付けを解除します。ハサミで軟部組織を慎重に取り外します。</li>
	<li>氷の上 α MEM の 2 mL の 6 ウェル プレートの各ウェルに 1 つ 1 つのマウスの遺伝子型のすべてのボーンを配置します。 注意: セル実行可能性を維持するために、骨は 1 時間未満をこれらの条件で保管すべき。</li>
</ol>3. 分離 (日-1)
<ol><li>Α MEM 培地 (3 mL) で 75% エタノール (3 mL) 6 ウェル プレートのウェル 1 個と他の 5 の井戸を埋めます。</li>
	<li>1 つの遺伝子型のすべてのボーンを入れて 5 回 10 α MEM 培地で 15 s と洗って骨のエタノール洗浄 s たびに。</li>
	<li>シザーと骨を断つ、50 mL の遠心管に α MEM 培地でのうち、骨の空洞とフラッシュの骨髄に 0.45 mm 注射針を挿入します。</li>
	<li>骨のもう一方の端から同じメソッドを使用して骨の空洞をフラッシュします。骨が薄いまで骨空洞を徹底的に洗うが、少なくとも 2 回繰り返します。</li>
	<li>遠心分離機の骨髄細胞ペレット 800 x g で 5 分吸引清 4 ° C を取得します。</li>
	<li>遠心分離機管と骨髄を再懸濁します優しくピペットに赤血球細胞の換散バッファーの 3 mL を加えます。赤い血液細胞を溶解する 8 分間氷の上遠心管を保ちます。</li>
	<li>セル換散を停止する遠心管に α MEM 媒体の 6 mL を追加します。500 x g と 10 分のための 4 ° C で遠心し、上清を吸引します。</li>
	<li>Α MEM 培地 3 mL に再懸濁します、6 ウェル プレート (ウェルあたり一般に 1 つのマウス) にセルを配置します。</li>
	<li>37 ° c 5% CO2インキュベーターで一晩インキュベートします。</li>
</ol>4. めっき (0 日)
<ol><li>未接続のセルに次の朝を収集するために 15 mL 遠心チューブに上清を転送します。</li>
	<li>800 x g と 5 分の 4 ° C で遠心し、上清を吸引します。</li>
	<li>Α MEM 培地 4 mL に再懸濁します。</li>
	<li>携帯ソリューションの 20 μ L を取るし、トリパン ブルーの 20 μ L で混ぜます。カウントの商工会議所に混合物の 10 μ L を追加し、ソリューションの mL あたりのセル数を取得します。</li>
	<li>Α MEM 培地 500,000 セル/mL のセル解を得るための適切な量を追加します。</li>
	<li>それぞれ 500 μ L と骨髄マクロファージ誘導培地の 50 μ L を 24 ウェル プレート、96 ウェル プレートの各ウェルに追加します。</li>
	<li>それぞれ 500 μ L とセル溶液 50 μ L を 24 ウェル プレート、96 ウェル プレートの各ウェルに追加します。</li>
	<li>3 日間、37 ° c 5% CO2インキュベーターで孵化させなさい。</li>
</ol>5. 分化 (日 3-7)
<ol><li>めっき後, 3 日 96 ウェル プレートの各ウェルから 50 μ L の培地を収集-20 ° C の凍結し、残留の培地を吸引します。</li>
	<li>それぞれ 1 mL と 100 μ L 破骨細胞誘導培地を 24 ウェル プレート、96 ウェル プレートの各ウェルに追加します。</li>
	<li>37 ° C で 2 日間インキュベートします。</li>
	<li>各ウェルから媒体の 50 μ L を収集し、残留の培地を吸引します。</li>
	<li>各ウェルに破骨細胞誘導培地を追加します。</li>
	<li>37 ° C で 1 日間インキュベートします。 注: この時点で大きい、多核巨細胞の破骨細胞は倒立顕微鏡 (図 2 a) の下で見てはなりません。</li>
	<li>破骨細胞は見ていない場合、は、破骨細胞誘導培地を変更し、破骨細胞が見られるまで毎日観察。</li>
	<li>破骨細胞が形成された後、96 ウェル プレートの各ウェルから媒体の 50 μ L を収集します。</li>
</ol>6. 酒石酸耐性酸フォスファターゼ (TRAP) 染色
注: 成熟破骨細胞は体外培養 (ステップ 4) の 6-7 日後にあります。
<ol><li>培地を吸引し、1x PBS で 3 回を優しく洗ってください。</li>
	<li>30 の修正プログラム ソリューションの 100 μ L の 96 ウェル プレートの各ウェルに、細胞を修正室温で s。</li>
	<li>固定後、修正プログラム ソリューションを吸引、prewarmed 37 ° c. に脱イオン水で 3 回を優しく洗浄脱イオン水を吸い出しなさい。</li>
	<li>トラップ汚れ 96 ウェル プレートの各ウェルに 100 μ L を追加し、37 ° C で 30 分と光からの盾のインキュベーターでプレートを配置します。</li>
	<li>汚損の後トラップ汚れを吸引、脱イオン水で 3 回を優しく洗います。</li>
	<li>イメージは、40 倍の倍率で倒立顕微鏡を用いた破骨細胞。</li>
</ol>7. トラップ活動定量化培地の
<ol><li>ステップ 5.1、5.4 5.8; から 96 ウェル プレートの各ウェルから 30 μ L の培養上清を収集します。陰性対照として非培養破骨細胞誘導培地の 30 μ L を使用します。新しい 96 ウェル プレートにサンプルを配置します。</li>
	<li>96 ウェル プレートの各ウェルに基板 mixture(1.5.5) の 90 μ L を追加します。</li>
	<li>1 h と光からの盾の 37 ° C でインキュベーターでプレートを配置します。</li>
	<li>96 ウェル プレートの各ウェルに 3 メートル naoh 水溶液 50 μ L の追加によって反作用を停止します。</li>
	<li>測定波長 405 theabsorbance nm。</li>
</ol>8. 西部にしみが付くこと
注: 6-7 日間 24 ウェル プレートで体外培養後成熟破骨細胞が表示されます。
<ol><li>培地を吸引します。</li>
	<li>予冷 1 x PBS 3 回で優しく洗ってください。</li>
	<li>PBS を吸い出しなさい。</li>
	<li>SDS 換散バッファー含むプロテアーゼ抑制剤のカクテル × 1 の 100 μ L を追加します。</li>
	<li>10 分 105 ° C で熱を可溶化物、12,000 × g と 4 ° C で 10 分間遠心するタンパク質を含む培養上清を収集します。</li>
	<li>10 %sds ページ11で蛋白質の 30 μ g を含む溶解液を分離し、ポリフッ化ビニリデン (PVDF) 膜に転送。</li>
	<li>30 分間 5% 乳脂肪の膜をブロックします。</li>
	<li>反猛禽、アンチ P S6、アンチ S6、抗 β-アクチン一晩 4 ° C で 5% 乳脂肪で縮尺希釈で膜を孵化させなさい。</li>
	<li>室温でトリス緩衝生理食塩水トゥイーン 20 (TBST) で 10 分間、膜を洗浄します。</li>
	<li>8.9 の手順を 2 回繰り返します。</li>
	<li>西洋ワサビペルオキシダーゼ (HRP) の膜を孵化させなさい-標識抗マウスやウサギの IgG 抗体 1:5,000 室温で 1 時間 5% 無脂肪牛乳で希釈。</li>
	<li>室温で 10 分間 TBST で 3 回膜を洗います。</li>
	<li>化学ルミネセンスの検出の膜に西洋化学発光 HRP 基板を追加し、室温で 5 分間インキュベートします。余分な基板のドレインし、x 線フィルムにしみを公開します。</li>
</ol>

<ol>
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すべての著者は、利害の対立があるないこと状態します。

Osteoclastogenic アッセイは、分離と培養破骨細胞の in vitro12,13最も広く使われている方法です。いくつかの RANKL による破骨細胞誘導説明13,14,15をされているが、本研究は、いくつかの変更は、以前の方法に基づくプロトコルを説明します。
以前の研究で?...

骨は絶えず変化する臓器で、骨芽細胞と破骨細胞は生涯で改造します。破骨細胞は、無機マトリックス吸収と骨芽細胞が合成し、新しいボーン行列1を分泌します。骨吸収と骨形成のバランスは骨の維持を含む骨の健康にとって重要な質量と刺激や損傷に応答します。このバランスを中断すると、骨粗鬆症と歯周疾患を含む、一連の骨代謝性疾患が発生します。これらの疾患では、破骨細胞性骨吸収による骨の質量損失は骨形成骨芽細胞2,3の容量を超えています。したがって、骨粗鬆症など骨格の疾患を治療する医薬品ターゲットを開発するために、世代や4破骨細胞の生物学を理解する重要です。
破骨細胞または骨の表面近くにあるユニークな巨大な多核巨細胞の細胞、単球/マクロファージ家族1に属する。イボットソン k. j.ら1, 25-ジヒドロキシ ビタミン D35を含む培地で培養破骨細胞様細胞を生成する手法について報告します。破骨細胞形成の本質的な要因としてマクロファージコロニー刺激因子 (M-CSF) と核因子 κ B ligand (RANKL) の受容体を活性化因子の同定には破骨細胞形成体外の効率が大幅に増加します。1,6,7. 世代の我々 の理解と破骨細胞の制御に培養破骨細胞の in vitroに能力は向上します。
2 つの構造的、機能的に異なる錯体、すなわち mTORC1、mTORC28,9ラパマイシン標的 (mTOR) 関数の哺乳類/機構対象。2 つの多蛋白質の複合体は異なった部品および下流の基板による互いから区別されます。mTORC1 には mTORC2 には mTOR (Rictor)9のラパマイシンを区別しないコンパニオンが含まれている mTOR (ラプター) のユニークな規制関連タンパク質が含まれています。mTORC1 は統合し、細胞の増殖、増殖と分化の調節に重要な信号を送信できます。最近、その mTORC1 破10で mTORC1 を不活化するラプターの削除によって異化骨吸収のネットワーク キー役割を果たしてデモンストレーションを行った。ただし、基になるメカニズムのさらなる研究が必要です。野生型 (WT) とラップのCtskマウスの骨髄由来マクロファージ (BMMs) から破骨細胞を生成し、破骨細胞に mTORC1 不活性化の影響を研究する本研究 RANKL ベースの osteoclastogenic メソッドを使用しました。形成します。

<table>
	<tbody>
		<tr>
			<td>Raptorfl/fl mice</td>
			<td>The Jackson Laboratory</td>
			<td>013188</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Corning</td>
			<td>10-022-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Gibico</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>BioInd</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse M-CSF protein</td>
			<td>R&#38;D</td>
			<td>Q3U4F9</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse RANKL protein</td>
			<td>R&#38;D</td>
			<td>Q3TWY5</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma-Aldrich</td>
			<td>302643</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate solution</td>
			<td>Sigma-Aldrich</td>
			<td>915</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Garnet GBC Base solution</td>
			<td>Sigma-Aldrich</td>
			<td>3872</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrite Solution</td>
			<td>Sigma-Aldrich</td>
			<td>914</td>
			<td></td>
		</tr>
		<tr>
			<td>Naphthol AS-BI Phosphate Solution</td>
			<td>Sigma-Aldrich</td>
			<td>3871</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate solution</td>
			<td>Sigma-Aldrich</td>
			<td>3863</td>
			<td></td>
		</tr>
		<tr>
			<td>Tartrate solution</td>
			<td>Sigma-Aldrich</td>
			<td>3873</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Corning</td>
			<td>21-031-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>L-tartaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251380</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tartrate dibasic dehydrate</td>
			<td>Sigma-Aldrich</td>
			<td>s4797</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase substrate</td>
			<td>Sigma-Aldrich</td>
			<td>P4744</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Raptor</td>
			<td>Cell Signaling Technology</td>
			<td>2280</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-P-ribosomal protein S6 (S235/236)</td>
			<td>Cell Signaling Technology</td>
			<td>2317</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ribosomal protein S6</td>
			<td>Cell Signaling Technology</td>
			<td>2211</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#946;-actin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-130300</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>Xilong scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polyvinylidene fluoride (PVDF) membrane</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Western Chemiluminescent HRP Substrate (ECL)</td>
			<td>Millipore</td>
			<td>00000367MSDS</td>
			<td></td>
		</tr>
		<tr>
			<td>IX71</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Envision</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.45-mm Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito forcep</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rankl-based osteoclast culture assay of mouse bone marrow to investigate the role of mtorc1 in osteoclast formation

破骨細胞が骨吸収をユニークな細胞を骨髄単球/マクロファージ系統から分化します。破骨細胞の機能不全、骨粗鬆症を含む骨代謝疾患のシリーズあります。病的骨の質量損失の防止のための医薬品のターゲットを開発するには、破骨細胞を前駆体から区別するメカニズムが理解されなければなりません。分離し、培養破骨細胞の多数の文化能力は破骨細胞分化における特定遺伝子の役割を決定するために重要です。破骨細胞の複雑な 1 (TORC1) ラパマイシンの哺乳類/機構のターゲットの不活化する破骨細胞数が低下し、骨の量を増やすただし、基になるメカニズムのさらなる研究が必要です。本研究では RANKL ベースのプロトコルを分離し、培養マウス骨髄から破骨細胞と破骨細胞形成における mTORC1 不活性化の影響を研究する、説明します。このプロトコルは正常に 1 週間以内通常巨大な破骨細胞の多数で起因しました。ラプターの削除は、破骨細胞形成を障害者し、その mTORC1 が破骨細胞形成のためクリティカルを示す分泌の酒石酸耐性酸性ホスファターゼの活性が低下します。

現在のプロトコルを使用して、巨大な破骨細胞の数が多いは、6; の日に見られました。巨大な破骨細胞を見ていない場合破骨細胞分化の 1 つのより多くの日があります (図 1) を必要とします。成功した破骨細胞形成は、TRAP 染色 (図 2 a) によって確認されました。破骨細胞以上 3 核の巨大ワイン赤/紫細胞はあった。WT BMMs (<s...

Watch this Scientific Journal Video about A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation at JoVE.com

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

本稿では、マウスの骨髄から破骨細胞形成の複雑な 1 ラパマイシンの哺乳類/機構のターゲットの役割を研究する分離と培養破骨細胞の in vitroにプロトコルについて説明します。

RANKL による破骨細胞文化アッセイのマウス骨髄破骨細胞形成における mTORC1 の役割を調査するには

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

a-rankl-based-osteoclast-culture-assay-mouse-bone-marrow-to

Research

JoVE Journal

Developmental Biology

12.7K ビュー.  National Clinical Research Center of Stomatology. 本稿では、マウスの骨髄から破骨細胞形成の複雑な 1 ラパマイシンの哺乳類/機構のターゲットの役割を研究する分離と培養破骨細胞の in vitroにプロトコルについて説明します。

ビデオ: A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

共培養系における神経結合の形成を評価するための光遺伝学的アプローチ

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

発生生物学

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酵素法は、凝固した骨髄試料からの間葉系幹細胞を救出する

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

の共焦点分析を用いました<em&gt;アフリカツメガエル</em&gt; WntおよびShhのモルフォゲン勾配の調節を調査するために、

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

ヒト骨髄から単離することMesangiogenic前駆細胞（MPCは）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

マウス胚中足骨の文化：軟骨内骨化の生理学的モデル

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

薬物治療と<em&gt;インビボ</em&gt;メダカ骨粗鬆症モデルにおける骨芽細胞破骨細胞の相互作用のイメージング

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

詳細な空間式マップからダイナミック発現データの時間的順序

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

マウスの破骨細胞前駆体におけるNotchシグナル伝達の刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

軟骨細胞分化のDNAメチル化レベルを定量化する5-mCドットブロットアッセイ<em&gt;インビトロ</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

マウスにおけるヒト化骨髄微小環境のバイオエンジニアリングとライブイメージングによるそれらの可視化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...