This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium &#8220;Tolerage&#8221;, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-W&#252;rttemberg.

本研究では、Regierungspräsidiumカールスルーエの倫理委員会によって承認されています。すべての動物は、ドイツ癌研究センター（DKFZ）で特定の病原体を含まない条件下で飼育しました。生後1~7日に至るまで、すべての培養実験の仔マウスのために使用しました。 胸腺からmTECsの1の単離注：以前に1滅菌条件下で説明したように、次のように次の消化工程はいくつかの変更を加えて実施しました。 <ol><li>仔マウスを刎ねると胸腺を削除します。 RPMI（5％FCSを含む）1640培地を含むペトリプレートに氷上で胸腺を置きます。 </li><li> 〜5〜30ミリリットルRPMI培地で丸底チューブに細かい、小さな作品や場所に胸腺をカットし、穏やかに室温で10分間、磁石を用いて撹拌します。 </li><li>その後、主に胸腺細胞を含む上清をデカントし、collagenaの1ラウンドで残りの組織を順次ダイジェスト37℃で25分ごとに、コラゲナーゼ/ディスパーゼ（0.2 mg / mlの1.2 U / mlの最終濃度）、続いて37℃でそれぞれ（0.2 mg / mlとし、最終concentartion 57 U / ml）で15分間SEタイプIV、胸腺まで磁気撹拌しながら水浴中でCは完全に消化されます。三つの胸腺への2つあたり1ミリリットルの酵素を使用してください。 </li><li>パスツールピペットで一度、7-10分の組織を攪拌します。コラゲナーゼ/ディスパーゼの画分をプールし、70ミクロンガーゼでろ過します。 </li><li>磁気細胞ソーティング（MACS）によりmTECsを豊か。以前11に記載され、 図1に示すように、磁気セルソーティングによってmTECsの精製を行います。 注：（1で使用し、G8.8：100希釈）および抗EpCAMバイオ：抗CD80-PE（100希釈16-10A1、1で使用します）mTECsの磁気選別のために、我々は、以下の抗体を使用した12。 MACSを使用して未成熟および成熟mTECsは次のように定義された： -のEpCAM + CD80 -及びCD45 - CD45 CD80 + respectivはエリー。 </li><li>以下に説明するようにMACS精製後（未熟mTECsの純度= 83.1±6.3％と成熟mTECs = 79.23±3.42パーセント）は、（2.3節）器官培養物にmTECsをシード。 </li><li>以下の抗体を用いて、CD45 MACS枯渇後（100ミクロンノズルを使用して）FACSによるあるいは、ソートmTECs：抗CD45-PerCPを（30-F11、1で使用：100希釈）、抗Ly51-FITC（6C3、1で使用：100希釈）、抗EpCAM-Alexa647（G8.8、1で使用：500希釈）および抗CD80-PE（16-10A1、1で使用：100希釈）。 （4日目）：ヨウ化プロピジウム（5,000 1）を使用して、死細胞を除外します。それぞれのEpCAM + CD80 + - CD45 - - Ly51 -のEpCAM + CD80 -およびPI - CD45 - Ly51 PI：FACSを使用して、未成熟および成熟mTECsは、次のように定義されました。 </li></ol> 2. 3D器官共培養（OTCs） 注：keratinocytの器官培養のための3D-真皮構築物先に説明したように9,13 ESを調製しました。すべてのステップで、細胞を37℃、5％CO 2でインキュベートしました。次のようにmTECsを使用してOTCsはわずかな変更を加えて調製しました。 <ol><li>ヒト線維芽細胞の調製 注：以前に9説明したように、ヒト皮膚線維芽細胞は、脱epidermised真皮の外植片培養物から得られました。 <ol><li>簡潔には、ヒトの皮膚（〜5cmの長さ）のストリップを切断し、サーモリシン、4℃でO / N（10mMのヘペス（pH7.4）で生理食塩水中の0.5mg / ml）で処理します。 </li><li>その後、ピンセットを用いて真皮から表皮を分離します。 </li><li>細かく10センチメートルペトリ皿に小片、所定の位置に真皮をカットし、無菌気流下で1〜2時間乾燥させます。 DMEM、20％FBSを含むと定期的に外植片を補います。 </li><li>外に成長している線維芽細胞を分割すると、コンフルエント植片は、さらに詐欺のためにプレートに付着し続けることを確認して、0.1％トリプシンを使用して（通常は約3週間）伸長のsecutiveラウンド。 </li><li> 14それらを、10％FBSを含むDMEMで最大3回、同じ外植片からの線維芽細胞を展開し、クライオ保ちます。各実験シリーズの線維芽細胞の同じバッチを使用してください。 注：線維芽細胞は、真皮同等物のセットアップのために照射されませんでした。 </li></ol></li><li>足場の調製<ol><li>正確に12ウェルフィルターインサートに適合するように鋭利な11ミリメートルの直径の金属パンチャーを使用して、よく画定円に0.4〜0.6ミリメートルの厚さのビスコース、不織繊維材料（Excelシートを支える製品の詳細を）カット。その後、足場として、12ウェルフィルターインサート（ポリエステル毛管細孔膜、3μmの孔サイズ）に配置します。滅菌12ウェルプレートに、完全なフィルタ設定を置きます。 </li><li>フィブリノーゲンおよびトロンビ​​ンの組み合わせからなる手術のためにフィブリン糊キットを使用してフィブリンゲルを準備します。フィブリノーゲンならびに8 mg / mlで、10のキットのトロンビン成分を事前に希釈それぞれ単位/ ml、。以下のように12ウェルプレートの1ウェル進行。 <ol><li> Ca 2+およびMg 2+、pH7.0のせずに、100μlのリン酸緩衝生理食塩水（PBS）を用いて100μlのフィブリノーゲン（8ミリグラム/ ml）を希釈します。 27万線維芽細胞を含む100μlのFCSで100μlのトロンビン（10単位/ ml）を希釈します。 </li><li>を2mg / mlのフィブリノーゲンおよび2.5単位/ mlのトロンビンの最終濃度で、その結果、（1混合物1）200μlのフィブリノゲンを追加している、足場上に細胞ミックス線維芽細胞を含むトロンビンの200μLを分注します。よく混ぜ、穏やかにピペッティング（1日目）によって足場の全域に​​わたって均等に分配します。 注：37℃で30分後、線維芽細胞を囲む血餅が完全に足場の内部空間を充填し、滑らかな上面を形成し、形成されているであろう。 </li></ol></li></ol></li><li> mTECsとの共培養<ol><li>前培養のために、器官培養を沈めますメディアの変化と、10％FBS、50μg/ mlのL-アスコルビン酸および1 ngの/ mlのTGF-β1を含むDMEM 4-5日間一日おきに。 </li><li> MTEC播種の日に、RFAD媒体によってメディアを交換します（1：1 DMEM + DMEM / F12）を、10％FBSを、10 -10 M、コレラ毒素、0.4 mg / mlのヒドロコルチゾン、50μg/ mlのL-アスコルビン酸、したがって、線維芽細胞によって分泌されるセリンプロテアーゼによって早熟線維素溶解を防止するRANKリガンド（を0.1μg/ ml）および500単位/ mlのアプロチニン、。 </li><li> 7-8時間後、セットアップ共培養を、線維芽細胞の足場の上にウェル当たり100μlの容量で25万mTECs（完全mTECs、またはCD80 LOおよびCD80 こんにちはサブセットのいずれか）を播種することにより。ノイバウアー室（4日目）を用いて細胞数を計測します。 注：胸腺3D器官培養物を空気持ち上げられる皮膚OTCsとは異なり、メディア内に浸漬されているすべての回で。 </li><li> 24時間のインキュベーションの後、今containi（ステップ2.3.2）上記のような培地で培養（総培地交換）を供給アプロチニン、250単位/ ml（5日目）の減少量をngの。 </li><li> mTECsの増殖活性を評価するために、培養の終了前4時間OTCsにEDU（6.7μM/ mlの、 すなわち、10μM/ウェル）を追加します。各培養標本のか流れによって2つのセクションでいずれかのカウントK14 + EDU +細胞による、mTECsの増殖指数を決定ケラチン14の共染色と組み合わせたエドゥイメージングキットに記載されているように店頭凍結切片の染色を行いますフローサイトメトリー（EDU 1.7節ではなくのLy51-FITCが1でCDR1-PB 15を使用し、フローサイトメトリー：100希釈し、2.3.6.4）。 </li><li>共培養の4-7日後、RNA分離、凍結切片またはFACS分析（日8-11）のためOTCs、処理を終了します。 <ol><li>ウェルインサートのフィルターから足場/真皮同等物を分離し、ピンセットを用いて培養を終了します。 </li><li>クライオセクショニングについては、OCT化合物で全体OTCを埋め込むとLiで凍結クライオセクショニング前に頭の良い窒素蒸気。使用するまで-20℃でクライオスタットとストアを使用して、5-7ミクロンの厚さのOTCのセクションを準備します。凍結切片OTCsの免疫組織化学（AF64 1で使用：1,000希釈）抗ケラチン14を使用してください：抗体、および抗ビメンチン（100希釈GP53、1で使用します）。それぞれの二次抗体を用いた間接免疫組織化学染色を行います。 </li><li> RNA単離のために、溶液2 mlのRNaseフリーのチューブのスクリューキャップで（フェノール及びチオシアン酸グアニジンを含む）を変性1ミリリットルを追加します。メスで小片に全体OTCをカットし、変性溶液を含むチューブに追加します。機械的には、（サンプルは、この時点の後に-80℃で保存することができる）2分間氷上の間に配置し、6.0の速度で30秒間回FASTPREP器具でOTCsを細断処理しました。 -80℃で凍結した場合は、氷上で解凍。 4℃で10分間、11,500-13,000 rpmでチューブを遠心。 RTおよびFで5分間インキュベートし、新しいチューブに上清を移し製造業者によって記載されるように酸性チオシアン酸グアニジニウム - フェノール - クロロホルム抽出を用いollow RNA単離プロトコール。 </li><li> FACS分析のために、足場を取り外し、フィブリン/線維芽細胞/ MTECゲルから膜を分離します。メスで細かく切断し、ゲルを〜20分間、または完全に磁気撹拌しながら水浴中で37℃にてコラゲナーゼ/ディスパーゼ2mlで消化されるまで、FACSチューブにそれを消化します。一度5分パスツールピペットを有する酵素液を攪拌します。完全消化した後、70μmのフィルターを通して細胞懸濁液をフィルタリングします。 1.7に記載のように抗体を用いて、単一細胞懸濁液を染色し、フローサイトメトリーによって分析します。 </li></ol></li></ol></li></ol>

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RTOCsと並んで、3D OTCsはTEC分化およびPGEのメンテナンス/誘導他（I）と比較した（ 表1）「単純化された3Dの文化」を使用しての面ではるかに優れていたことで-線維芽細胞を、単独で足場なし。二次元システムを用いて-のTEC 10と線維芽細胞/フィーダー細胞を共培養し、TECのクローンの開発が、PGEが失われた式（III）3T3-J2細胞、（IV）マトリゲルまたは（V）ECM成分（未発表?...

残りの2％を一括胸腺間質（ すなわち、上皮細胞、樹状細胞、マクロファージ、B細胞、線維芽細胞、内皮細胞）を構成する種々の細胞で構成されていながら、開発胸腺細胞は、胸腺の約98％を占めています。外側皮質上皮細胞（cTECs）は、骨髄、多能プレT細胞におけるT細胞系譜の誘導と自己MHCのポジティブ選択からプロT細胞の移民を調達未熟胸腺細胞を制限しました。内側の髄質胸腺上皮細胞（mTECs）はいずれかの誘導陰性選択またはT調節細胞系譜へのそれらの偏差による自己ペプチド/ MHC複合体のための高親和性TCRを有するもの胸腺細胞の寛容の誘導に関与しています。中央寛容誘導のコンテキストでは、mTECsは、彼らがこのように周辺自己ミラーリング組織限定自己抗原（TRAS）の広いスペクトルを表現するという点でユニークです。この現象は、無差別遺伝子発現（PGE）と呼ばれています1,2。 様々な短期の2D培養システムは、必ず最初の2日3-6内PGEおよびMHCクラスII、FoxN1とアイレのような重要な調節分子の喪失をもたらしたように、この魅力的な細胞型の最新の研究は、ex vivoで単離された細胞に依存しています。これは、特定の成分および胸腺の無傷の3次元網目構造の特徴は、2次元モデルで欠落した、しかし不明でした。再凝集胸腺器官培養（RTOC）は無傷の胸腺の微小環境7に、他方では、これまでのところ唯一の3D一方で、T細胞の発達の研究を可能にするシステム、および間質細胞生物学でした。しかし、RTOCsは一定の制限がある、 すなわち、それらは既に、細胞の複合混合物を含む、胎児間質細胞の入力を必要とし、5〜10日の最大の培養期間に耐えます。 体外培養系における還元主義の欠如は、の研究を妨げていますT細胞の発達と胸腺器官形成のいくつかの側面はなく、少なくとも分子PGEの調節およびmTECsの発生生物学との関係。 皮膚および胸腺の上皮細胞の構造化された組織の近接関連性のために、我々は、in vitroでケラチノサイトの分化をエミュレートするため、真皮等価物を作成するために独自に開発された3D器官培養（OTC）のシステムを選択しました。 OTCシステムは、ケラチノサイトが8,9を播種し、その上にフィブリンゲル中に閉じ込められている真皮線維芽細胞を重層不活性足場マトリックスで構成されています。ここでは、精製されたmTECsでケラチノサイトを置き換えます。このモデルの基本的な機能を維持しながら、私たちは、特定のパラメータを最適化します。 採用店頭モデルmTECsでは、増殖分化を受け、MTECのアイデンティティとPGEを維持し、このように密接にインビボ mTECs開発の模倣<sup&gt; 10。このテクニカルノートでは、胸腺OTCsの段階的なセットアップを可能にする詳細なプロトコルを提供します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 mice&#160;</td>
			<td>Charles River WIGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Microbeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (G8.8 -Alexa 647 and -biotin)</td>
			<td>Ref. 12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD80-PE antibody</td>
			<td>BD Pharmingen</td>
			<td>553769</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-PerCP antibody</td>
			<td>BD Pharmingen</td>
			<td>557235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly51-FITC antibody</td>
			<td>BD Pharmingen</td>
			<td>553160</td>
			<td></td>
		</tr>
		<tr>
			<td>CDR1-Pacific Blue</td>
			<td>Ref. 15</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 14 antibody</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin antibody</td>
			<td>Progen</td>
			<td>GP58</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated AffiniPure F(ab&#39;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>106-546-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Molecular Probes (Invitrogen GmbH)</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">Click-iT EdU Alexa Fluor 594 Imaging Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">C10339</a></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">C10425</a></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well filter inserts (thincerts)</td>
			<td>Greiner bio-one</td>
			<td>657631</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Greiner</td>
			<td>665180-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Jettex 2005/45</td>
			<td>ORSA, Giorla Minore, Italy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Serva</td>
			<td>47302.03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Lonza</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-049&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Gold</td>
			<td>GE Healthcare</td>
			<td>A11-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin (Trasylol)</td>
			<td>Bayer</td>
			<td>4032037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Biomol</td>
			<td>G117</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Seromed (Biochrom)</td>
			<td>K3520</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#223;1</td>
			<td>Invitrogen</td>
			<td>PHG9214</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>R&#38;D systems</td>
			<td>462-TR-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysin</td>
			<td>Sigma Aldrich</td>
			<td>&#160;T-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>TissueTek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (aka. Denaturing solution - Acid guanidinium thiocyanate-phenol-chloroform extraction)</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		</tr>
		<tr>
			<td>FastPrep FP120</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV&#160;</td>
			<td>CellSystems</td>
			<td>LS004189</td>
			<td>0.2 mg/ml and 57U/ml final conc.</td>
		</tr>
		<tr>
			<td>Neutrale Protease (Dispase)</td>
			<td>CellSystems</td>
			<td>LS002104</td>
			<td>0.2 mg/ml and 1.2U/ml final conc.</td>
		</tr>
		<tr>
			<td>DNase I&#160;</td>
			<td>Roche</td>
			<td>11 284 932 001</td>
			<td>25 &#181;g/ml final conc.</td>
		</tr>
	</tbody>
</table>

3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression

イントラ胸腺T細胞の発達は、様々な間質細胞、 すなわち、非T細胞から構成される複雑な三次元網目構造を必要とします。胸腺細胞は、末梢に先立ってエクスポートに、順次義務のチェックポイントを通過しながら、高度に調整された時間的、空間的順序でT細胞受容体レパートリーの生成と選択に続いて、すなわち、T細胞系列のコミットメントを、この足場を横断します。この足場を形成する2つの主要な居住細胞型（cTECs）皮質及び髄質胸腺上皮細胞（mTECs）です。 mTECsの重要な特徴は、多数の組織限定抗原のいわゆる無差別式です。これらの組織制限抗原は、それぞれ、自己寛容を生じ、mTECsまたは胸腺樹状細胞によって直接的または間接的に胸腺細胞の未成熟するために提示されます。 cTECsとmTECsの発生経路と機能をエミュレートし、in vitroでの適切なモデルは、現在、LACですキング。適切な実験モデルの欠如は、例えば、まだ不十分な細胞および分子レベルで理解されて無差別遺伝子発現の分析を妨げました。我々は、単離されたmTECs ex vivoで培養するために3Dの器官共培養モデルを適応しました。このモデルは、もともとインビトロでの皮膚同等物を生成するような方法で、ケラチノサイトを培養するために考案されました。 、CD80 こんにちは 、アイレ陽性mTECs、RANKLへの（ii）の反応性、およびFoxN1の（iii）の持続的な式に（I）の増殖およびCD80 LO、アイレ陰性の分化：3DモデルはMTEC生物学の重要な機能的特徴を保存しますアイレとCD80 こんにちは mTECsで組織限定遺伝子。

私たちは、もともとケラチノサイト9 のインビトロ長期培養のために開発された3D器官の共培養モデル（3D OTC）を採用しました。 MACS富化mTECsは（MACS濃縮スキーム図1参照）フィブリンゲルおよび封入された線維芽細胞からなる足場に播種しました。線維芽細胞は、 インビトロで mTECsをサポートする必須の細胞外マトリックス（ECM）を提供します。 MTECsは（OTC?...

Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

髄質胸腺上皮細胞の増殖、分化および無差別遺伝子発現を支える3D器官共培養モデル

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

3d-organotypic-co-culture-model-supporting-medullary-thymic

Research

JoVE Journal

Developmental Biology

10.6K ビュー.  German Cancer Research Center (DKFZ). Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression. 

ビデオ: 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化と共培養マウスの卵巣と卵胞

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

発生生物学

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

レチノイン酸誘導された胚性幹細胞分化の間に活性化された予測と遺伝子調節要素の検証

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

糖尿病性角膜症のためのアデノウイルス遺伝子治療：ヒト臓器培養角膜と​​輪部上皮細胞における創傷治癒に及ぼす影響と細胞マーカーの発現を幹

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

オプションの 2 D 加工で合理化された 3 D 小脳分化プロトコル

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

並べ替え内皮細胞ニッチ共培養と組み合わせて 1 つのセル インデックスを使用して胚性造血幹細胞前駆体のクローン解析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

マウス骨髄前駆体 32D/G-CSF-R の増殖・分化細胞

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

転写因子の強制発現によるヒト線維芽細胞のヘモジェニックリプログラミング

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

生体内でのマクロファージ募集をテストするための異所性ケモカイン発現モデル

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...