この作品は、デットFrieForskningsråd/ Sundhed OG SygdomからNCCRフロンティア遺伝学におけるパイロット賞、若年性糖尿病研究財団助成41-2009-775とグラント12から126875で順次資金を供給された。著者らは、動画撮影をホストするためのスパニョーリラボに感謝。

このプロトコルは、マウスE10.5から派生した膵臓オルガノイドが上皮膵臓細胞を解離した成長を目指しています。 プロトコルは、動物実験のための倫理的な承認を必要とする。 E10.5マウス胚から背膵芽の1。解剖<ol><li>ハサミで腹部を開き、胎生（E）10.5で時限妊娠マウスを犠牲に、2子宮角を削除し、冷リン酸緩衝生理食塩水（PBS）、またはダルベッコで満たされた10センチメートルペトリ皿に置いて修正必須培地（DMEM）は氷上に保持。細胞播種の犠牲からの全実験は、細胞の損傷を防ぐために60〜90分間で行われる。 </li><li>小さなハサミを使用して個々の胚のセグメントに子宮を分離する。冷PBSで35ミリメートルペトリ皿に1胚を移し、上からの照明で解剖顕微鏡下でそれを視覚化する。解剖ピンセットで周囲の筋肉、脱落膜と卵黄のSAを削除Cおよび胚を公開します。背中冷たいDMEM培地に胚を置きます。胚は容易に3ミリリットルのプラスチックトランスファーピペット/スポイトを使用して転送することができる。続行する前に、同様の方法で、すべての個々の胚を分離します。 </li><li> PBS中できれいな35ミリメートルペトリ皿に胚を置きます。 <ol><li>前肢を削除するために、薄い鉗子（0.05ミリメートル幅）を使用してください。優しく脊髄領域（ 図1）からの消化管を分離するために開口部に鉗子を挿入します。オプション：より便利に、胃を参照してください下に卵黄茎下の心臓や尾部に、胚の上半身を削除するには。 </li><li>胃、肝臓と腸の位置を確認します。鉗子を使用すると、胃から小腸への消化管に分離した（ 図1）氷の上に冷たいDMEM中に入れてください。背側膵臓芽は、胃への後方（ 図1）、背側に取り付けられている。 </li><li>シミのすべての個々の消化管を隔離続行する前にLAR方法。胚が遺伝子型を決定する必要がある場合には、解剖時に尾を収集し、氷上で冷たいDMEMで24ウェルプレート中の異なるウェル内のすべての個々の消化管を保つ。 </li></ol></li><li>冷PBS中できれいな35ミリメートルペトリ皿に1消化管を配置します。今解剖顕微鏡下で背側膵臓芽を視覚化して分析する明視野で下から照明を使用しています。これらの条件はボリュームの可視化を最適化します。電解的にシャープタングステン針または20gの注射針を使用して、上皮の周り可能な限り少ない間充織（ 図1）と膵臓芽を分離します。 <ol><li> 2〜3分間冷たいディスパーゼ溶液（1.25ミリグラム/ ml）を含むペトリ皿に孤立した芽を転送します。この時点から、移動が最も便利に口の制御、フレキシブルチューブに付着した難引っ張っ50μlのガラスキャピラリーで行うことができます。代わりに、より多くのRIと芽を失うSK、適切なプラスチックのヒントを10μlの自動ピペット（ピペットマン）を使用します。 </li><li>微視的な制御の下で膵臓芽の吸引と排出を行う。バックPBS中の膵臓芽を置く。さらにガラスキャピラリー（ 図1）を使用して、針と穏やかな吸引との間葉から分離された膵臓の芽を清掃してください。 </li><li>全体間充織が除去されると、冷PBS中で膵臓芽をすすぐ。個々のウェル（冷DMEM中の10μLで満たされた60ウェルミニトレー）冷DMEM中に各芽を移す。それは組織が非常に粘着性になり、これディスパーゼで間充織を削除しないことが重要です。 </li></ol></li></ol>分散した細胞の2。メッキと文化<ol><li>洗浄に10μlのPBSで満たし60ウェルミニトレーの円錐形のウェルに難引っ張らガラスキャピラリーを持つすべての胚から解剖上皮を転送します。 <ol><li>トリプシン0.05％Aを10μlに芽を移すNDそれは4分間、37℃でインキュベートしてみましょう。 10μlのDMEM + 10％ウシ胎児血清（FCS）でウェルに芽を転送することによりトリプシンを不活性化する。 </li><li>ピペットプラーで引っ張ら細い毛細血管を通して吸引により細胞懸濁液を解離する。これは、細胞を解離するには、上下にピペッティングしながら、この段階で泡を避けることが重要である。膵臓オルガノイド最適に5月15日細胞の小グループから始めるため、部分的な解離（ 図1）をお勧めします。 </li></ol></li><li>個々の処理による違いを最小限にするために、エッペンドルフチュー​​ブに、いくつかの胚からの細胞をプールする。 1:3の比で冷却したマトリゲル中の細胞懸濁液を希釈する。分量この混合物を96ウェルプレートに、8μL/ウェルまたは画像化のために最適化されたプレート（下記参照）。 </li><li>マトリゲル増粘させること、5分間37℃でプレートをインキュベートする。 タブを参照して、選択（オルガノイドや球の培地を70μlの井戸を埋めるレ1および2）、37℃、5％CO 2及び95％空気を含む加湿環境ではあり</li><li>すべての4 日目の媒体を交換してください。毎日成長している膵臓コロニーを監視し、画像化してプロセスを文書化します。 </li><li> 28先に報告されたように関心対象の小分子またはタンパク質は、干渉実験のために、この段階で培地に添加することができる。 </li></ol> 表1：オルガノイドミディアム。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>素材の名前</td><td>ストック濃度</td><td>最終的な培地中の濃度</td><td>株式のボリューム</td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td> 100パーセント</td><td> 1％ </td><td> 50μL </td></tr><tr><td>ノックアウト血清代替（サプリメント） </td><td> 100パーセント</td><td> 10パーセント</td><td> 500μL </td></tr><tr><td> 2 - メルカプトエタノール</td><td> 14.3 M </TD&gt; <td> 0.1 mMの</td><td> 1μL </td></tr><tr><td>ホルボールミリステートアセテート（PMA） </td><td> 16μM </td><td> 16 nMの</td><td> 5μL </td></tr><tr><td> Y-27632（ROCK阻害剤） </td><td> 50 mMの</td><td> 10μMの</td><td> 1μL </td></tr><tr><td> EGF </td><td> 50μg/ mlの</td><td> 25 ng / mlの</td><td> 2.5μL </td></tr><tr><td>組換えヒトR-スポンジン1 </td><td> 250μg/ mlの</td><td> 500 ng / mlの</td><td> 10μL </td></tr><tr><td> - または - </td><td></td><td></td><td></td></tr><tr><td>組み換えマウスR-スポンジン1 </td><td> 250μg/ mlの</td><td> 500 ng / mlの</td><td> 10μL </td></tr><tr><td>組換えヒトFGF1（aFGFを） </td><td> 100μg/ mlの</td><td> 25μg/ mlの</td><td> 1.25μL </td></tr><tr><td>ヘパリン（Liquemin） </td><td> 2500 U / mlの</td><td> 2.5 U / mlの</td><td> 2μL </td></tr><tr><td>推奨遺伝子組換え型のヒトFGF10 </td><td> 100μg/ mlの</td><td> 100 ng / mlの</td><td> 5μL </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25μL </td></tr><tr><td>合計</td><td></td><td></td><td> 5000μL </td></tr></tbody></table> 表2：スフィア媒体。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>素材の名前</td><td>ストック濃度</td><td>最終的な培地中の濃度</td><td>株式のボリューム</td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td> 100パーセント</td><td> 1％ </td><td> 50μL </td></tr><tr><td> B27のX50（サプリメント） </td><td> 100パーセント</td><td> 10パーセント</td><td> 100μL </td></tr><tr><td>組換えヒトFGF2（bFGFを） </td><td> 100μg/ mlの</td><td> 64 ng / mlの</td><td> 3.2μL </td></tr><tr><td> Y-27632（ROCK阻害剤） </td><td> 50 mMの</td><td> 10μMの</td><td> 1μL </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845.8μL </td></tr><tr><td>合計</td><td></td><td></td><td> 5000μL </td></tr></tbody></table>オルガノイド開発の進行の3。イメージング<ol><li>画像オルガノイドどちら毎日または時間経過顕微鏡による蛍光タイムラプス顕微鏡を使用。タイムラプスイメージングのため、XY（Z）自動化された倒立蛍光顕微鏡を使用しています。 </li><li> 4ウェルプレートまたは2〜5ミリリットル培地を充填したガラス底プレート中3μLの預金小滴。 10倍の長距離目的としたイメージ。注：蛍光トレーサーを発現するトランスジェニックマウスを使用することができる。例えば、映画1は、PDX1のNgn3-ER TM-IRES-nGFP +マウス4からオルガノイドの初期膨張。核GFPは、個々のオブジェクトが、同様の原理は、膜の蛍光を有する細胞を追跡するために適用することができるように、細胞を追跡することを可能にするMT / Mg系マウス29など。 </li><li>フォーカスドリフトを回避し、手動で定義されている位置で、3日以上1画像/時を取るために自動化を制御するソフトウェアを設定するために細胞を播種した後タイムラプスイメージングの3時間後に開始します。全ての位置については、蛍光マーカーの発現を報告し、微分干渉コントラスト（DIC）画像ならびにGFP信号を取得する。 </li></ol>組織学のためオルガノイドの4。回収<ol><li>氷上で96ウェルプレートを置き、氷冷PBSで置き換え、培地を除去。これは部分的にマトリゲルを解重合する。 </li><li>優しくアーキテクチャ全体を混乱させないために千μLチップを使用して、周囲のマトリゲルを除去し、個々のオルガノイドを吸引する。氷冷PBSでウェルに各オルガノイドを転送します。氷の上にプレートを保管してください。マトリゲル内直接固定することも可能である。 </li><li> 、4％パラホルムアルデヒド（PFA）で15分間オルガノイドを修正しました。ショ糖でそれを冷凍保存し、ゼラチンに埋め込む。それぞれを処理前述したように凍結切片および組織学のための組織体（ヨハンソンら 、2007）4。 </li></ol> PCRおよび生化学用オルガノイドの5。回復<ol><li>氷上で96ウェルプレートを配置し、培地を除去。細胞のRNAを安定化し、保護するために、ウェル当たりのRNAlater 60を添加する。 </li><li>部分的に氷の上で解重合することにより、各ウェル内でゲルを機械的に破壊する。全体的なアーキテクチャを混乱させるか、200μlの先端を機械的にゲルを破壊することによって（マトリゲルで）全体をよく回復しないために、千μLチップを使用して個々のオルガノイドを回復のどちらか。 RNA分解酵素フリー非粘着性エッペンドルフチュー​​ブに入ってもコンテンツを転送するために千μlのチップを使用して、氷上に保持。 </li><li> 60μLのRNAlaterでウェルを洗浄し、同じエッペンドルフチュー​​ブに残りのコンテンツを追加します。 </li><li> 4℃で500〜1,000×gで5分間チューブをスピン</li><li> 20〜30μLを残して上清を取り除き一緒にペレットとチューブ内のRNAlater。生化学のために、可能な限りドライサンプルを格納。 </li><li>すぐにRNAlater中でサンプルを凍結しないでください。 4℃のCO / Nにおけるストア（RNAlaterで徹底的に組織を貫通できるようにする）。組織は、長期保存のために-20℃で保存することができ、後に組織破壊及びRNAの少量の抽出のために処理することができる。 </li></ol>

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U., Goldstein, A. S., Lawson, D. A., Cheng, D., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation+and+characterization+of+adult+murine+prostate+stem+cells.">Isolation, cultivation and characterization of adult murine prostate stem cells.</a> Nat Protoc. 5, 702-713 (2010).</li><li>Rock, J. R., 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=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proc Natl Acad Sci U S A. 106, 12771-12775 (2009).</li><li>Eiraku, 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Spence, J. R., 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=Directed+differentiation+of+human+pluripotent+stem+cells+into+intestinal+tissue+in+vitro.">Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.</a> Nature. 470, 105-109 (2011).</li><li>Lancaster, M. A., 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 10, (2013).</li><li>Greggio, C., 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=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

The authors have nothing to disclose.

インビトロにおける機能的β細胞の大規模生産は依然として1無効である。このような厳しい状況では、発生生物学の研究で​​は、機能的なβ細胞の分化に必要とされる正確な信号を解読するに役立つことがあります。このプロトコルは、in vitroでの胚性膵臓前駆細胞の維持、拡大と差別化が可能になります。これは、他の内分泌ホルモンを共発現しないインスリン産生...

器官培養は複雑であるが、生体内の調査で非常に関連性の高い細胞株モデルの便利なが、おおよそのシミュレーション間のギャップを橋渡しする有用なモデルを提供しています。内分泌および外分泌細胞を模擬した形質転換された細胞株が存在するが、膵臓例では、膵臓の前駆細胞と完全に同等のない細胞株ではありません。大人の膵臓全体を培養することができません。単離された内分泌膵島は数時間5 インビトロで維持することができる細胞増殖および組織切片なしで、数週間維持することができる。胚性膵臓培養物は、広くその発達を研究するためだけに使用されているだけでなく、上皮間葉相互作用4,6,7を調査するために、画像に8を処理または化学的にそれらと9を妨害する。二つの器官培養法が主に使用されます。最初は都合のですフィブロネクチンコーティングしたプレート2、上の膵臓芽を培養することにある画像化目的のためにnient;二つ目のオプションは文化に最も形態形成を保持する気液界面3,4でフィルタの臓器です。非常に有用であるが、これらの方法は、ある程度の平坦化をもたらす;前駆細胞の拡大は非常に正常な発達および出発集団は、膵臓細胞および間葉細胞の全ての種類を含む複合体であると比べて制限される。 文化への能力と分散初代細胞を拡大し、系統関係を研究し、単離された細胞のタイプ10の固有の特性を明らかに価値がある。杉山ら 11膵臓前駆細胞およびフィーダー層上の文化の中で3〜5日間いくつかの機能の文字を保持する内分泌前駆細胞を維持することができます。ニューロスフェア12と13のマンモスフィアに似Pancreatospheresは、成人の島および前駆細胞/幹細胞の性質であるが管細胞から展開されてきたこれらの球体を生成することをすることは明らかではない。また、生理的発達とは対照的に、pancreatospheresは、いくつかのニューロンの14,15を含んでいた。球はまた、最近、胚性膵臓前駆細胞16,17と良好な前駆細胞の拡大とそれに続く分化と膵臓18回生から生じたが、形態形成を再現できなかったし。 小型化された臓器に自己組織化分散させ、多くの場合、定義された細胞からの3Dモデルは、最近、繁栄し、そのような腸19,20、胃21、肝臓22、前立腺23、気管などの複数の器官の発達、大人の離職率をシミュレートしている24。光学カップ25、小腸26または脳27の場合のように、いくつかの例では、形態形成の発達および分化は、ES細胞から3Dにで要約されている。 ここでは、DES彼らは差別化と自己組織化することができる3Dマトリゲル足場に解離した多能性膵臓前駆細胞を拡大する方法をcribe。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
			<td>M0815-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

3Dマトリゲルで解離し、播種E10.5の背側膵臓前駆細胞は、膵臓の発達を再現。前駆細胞は、最も容易に蛍光リポーターで追跡することができる。我々のケースでは（ 図2）NEUROG3 4を活性化することなく、タモキシフェンの非存在下では、したがって、Pdx1のプロモーター（、PDX1のNgn3-ER TM-nGFP）（動画1）によって制御された核GFPタンパク質を発現するトランス...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

このプロトコルで説明した3次元培養法は、分枝状の臓器へのそれらの大幅な拡大、分化および形態形成などの分散したマウス胎児の膵臓前駆細胞から膵臓の発達を再現。この方法は、画像化、ニッチの機能的干渉や操作に適している。

分散マウス胚性前駆細胞からin vitro膵臓器官形成

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

in-vitro-pancreas-organogenesis-from-dispersed-mouse-embryonic

Research

JoVE Journal

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K ビュー.  Swiss Institute for Experimental Cancer Research. このプロトコルで説明した3次元培養法は、分枝状の臓器へのそれらの大幅な拡大、分化および形態形成などの分散したマウス胎児の膵臓前駆細胞から膵臓の発達を再現。この方法は、画像化、ニッチの機能的干渉や操作に適している。 

ビデオ: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

ハンギングドロップ法を用いたマウス胚性幹（MES）細胞のin vitroにおける分化

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

生物学

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

磁気共鳴イメージングのためのヒト胚性幹細胞のin vitroでラベリングの

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

骨格筋からの前駆体の精製

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

マウス胎児心臓の開発に細胞標識と注入

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

マウス膵臓からのRNAの単離：リボヌクレアーゼが豊富な組織

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

途上胚性マウス心臓弁地域からのタンパク質の単離

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...