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.

Questo studio è stato approvato dal comitato etico del Regierungspräsidium Karlsruhe. Tutti gli animali sono stati alloggiati in condizioni esenti da organismi patogeni specifici presso il Cancer Research Center tedesco (DKFZ). Per tutti i cuccioli di esperimenti di coltura del mouse che vanno da 1 a 7 giorni di età sono stati utilizzati. 1. Isolamento di mTECs da Timo NOTA: Le seguenti operazioni di digestione sono state eseguite come descritto in precedenza 1 in condizioni sterili con alcune modifiche come segue. <ol><li> Decapitare i cuccioli di topo e rimuovere il timo. Posizionare il thymi su ghiaccio in una piastra di Petri contenenti terreno RPMI 1640 (contenente 5% FCS). </li><li> Tagliare il thymi in sottili, piccoli pezzi e posto in una provetta a fondo tondo con ~ 5-30 ml RPMI media e mescolate delicatamente con un magnete per 10 minuti a temperatura ambiente. </li><li> In seguito, decantare il surnatante contenente principalmente timociti e digerire la sequenza tessuto rimanente con un giro di collagenasé di tipo IV (0,2 mg / ml e 57 U / ml concentartion finale) per 15 minuti ciascuno a 37 ° C, seguita da collagenasi / dispasi (0,2 mg / ml e la concentrazione finale 1,2 U / ml) per 25 min a 37 ° ciascun C in un bagno d&#39;acqua con agitazione magnetica fino alla thymi sono completamente digerito. Usare 1 ml enzimatica per 2-3 thymi. </li><li> Agitare il tessuto volta ogni 7-10 minuti con una pipetta Pasteur. Si riuniscono gli collagenasi / frazioni dispasi e filtrare attraverso una garza di 70 micron. </li><li> Arricchire i mTECs da cellule magnetico ordinamento (MACS). Eseguire la purificazione di mTECs da cellule magnetico classificare come descritto in precedenza 11, e mostrato in Figura 1. NOTA: Per l&#39;ordinamento magnetico di mTECs abbiamo usato i seguenti anticorpi: anti-CD80-PE (16-10A1, uso a diluizione 1: 100) e anti-EpCAM-bio (G8.8, uso a diluizione 1: 100) 12. MTECs immaturi e maturi con MACS sono stati definiti come: CD45 - EpCAM + CD80 - e CD45 - CD80 + respectively. </li><li> Dopo MACS purificazione (purezza del mTECs immaturi = 83,1 ± 6,3% e mTECs maturi = 79.23 ± 3,42%), seminare i mTECs sulle colture organotipiche come descritto di seguito (punto 2.3). </li><li> In alternativa, ordinare mTECs di FACS (utilizzando 100 ugelli micron) dopo CD45 MACS esaurimento utilizzando i seguenti anticorpi: anti-CD45-PerCP (30-F11, l&#39;uso in diluizione 1: 100), anti-Ly51-FITC (6C3, utilizzare a 1 : 100 diluizione), anti-EpCAM-Alexa647 (G8.8, uso in diluizione 1: 500) e l&#39;uso anti-CD80-PE (16-10A1, a diluizione 1: 100). Escludere le cellule morte con ioduro di propidio (1: 5.000) (4 ° giorno). MTECs immaturi e maturi utilizzando FACS sono stati definiti come: PI - CD45 - Ly51 - EpCAM + CD80 - e PI - CD45 - Ly51 - EpCAM + CD80 +, rispettivamente. </li></ol> 2. 3D organotipiche co-culture (OTC) NOTA: I costrutti 3D-cutanea per la coltivazione organotipica di keratinocytes state preparate come descritto in precedenza 9,13. In tutte le fasi cellule sono state incubate a 37 ° C e 5% CO 2. I farmaci da banco che utilizzano mTECs sono stati preparati con lievi modifiche nel modo seguente. <ol><li> Preparazione di fibroblasti umani NOTA: i fibroblasti dermici umani sono stati ottenuti da colture espianto di de-epidermised derma come descritto in precedenza 9. <ol><li> In breve, tagliare le strisce di pelle umana (~ 5 cm di lunghezza) e trattare con termolisina (0,5 mg / ml in soluzione salina con 10 mM Hepes pH 7,4) O / N a 4 ° C. </li><li> Successivamente, separare l&#39;epidermide dal derma con pinze. </li><li> Tagliare finemente il derma in piccoli pezzi, posto in un piatto 10 centimetri Petri e lasciare asciugare per 1-2 ore sotto un flusso d&#39;aria sterile. Supplemento espianti regolarmente con DMEM contenente 20% FBS. </li><li> Dividere i fibroblasti crescita fuori quando confluenti (di solito circa 3 settimane) con 0,1% tripsina fare in modo che gli espianti continuano ad aderire alla piastra per ulteriori conturni consecutivi di conseguenza. </li><li> Espandere i fibroblasti dalle stesse espianti per un massimo di 3 volte in DMEM con 10% FBS e crio-conservare le 14. Utilizzare lo stesso lotto di fibroblasti per ogni serie sperimentale. NOTA: I fibroblasti non sono stati irradiati per il set-up degli equivalenti dermici. </li></ol></li><li> Preparazione del impalcatura <ol><li> Tagliare i 0,4-0,6 mm viscosa spessore, materiale fibroso non tessuto (dati del prodotto a sostegno foglio Excel) in ambienti ben delimitate con un netto 11 millimetri di diametro puncher metallo per adattarsi esattamente in 12 inserti bene col filtro. Poi inserirla nel pozzo 12 inserto del filtro (poliestere membrana capillare pori, 3 micron dimensione dei pori) da impalcatura. Posizionare la configurazione filtro completo in un 12-pozzetti sterili. </li><li> Preparare il gel di fibrina utilizzando una colla di fibrina-kit per la chirurgia costituito da una combinazione di fibrinogeno e trombina. Pre-diluire il fibrinogeno e la trombina-componente del kit a 8 mg / ml e 10Unità / ml, rispettivamente. Per un singolo pozzetto di una piastra 12 pozzetti procedere come segue. <ol><li> Diluire 100 microlitri fibrinogeno (8 mg / ml) con 100 ml di tampone fosfato salino (PBS) senza Ca 2 + e Mg 2 +, pH 7,0. Diluire 100 ml di trombina (10 unità / ml) con 100 microlitri FCS contenenti 270.000 fibroblasti. </li><li> Dispensare 200 microlitri della trombina contenente fibroblasti cellule preconfezionato sul ponteggio, a cui si aggiungono 200 pl fibrinogeno (miscela 1: 1), ottenendo una concentrazione finale di fibrinogeno di 2 mg / ml e di trombina di 2,5 unità / ml. Mescolare bene e distribuire in modo uniforme su tutta l&#39;area del ponteggio pipettando dolce (giorno 1). NOTA: Dopo 30 minuti a 37 ° C un coagulo che racchiude i fibroblasti sarà formata, riempiendo completamente gli spazi interni del ponteggio e formando una superficie superiore liscia. </li></ol></li></ol></li><li> Co-coltura con mTECs <ol><li> Per la pre-cultura, immergere le colture d&#39;organo aDMEM con 10% FBS, 50 mg / ml di acido L-ascorbico e 1 ng / ml di TGF-β1 con una variazione media ogni altro giorno per 4-5 giorni. </li><li> Il giorno della MTEC semina, sostituire il mezzo attraverso RFAD media (1: 1 DMEM + DMEM / F12) con 10% FBS, 10 -10 M tossina colerica, 0,4 mg / ml idrocortisone, 50 mg / ml di acido L-ascorbico, RANK ligando (0,1 mcg / ml) e 500 unità / ml di aprotinina, impedendo così fibrinolisi precoce da serina proteasi secrete da fibroblasti. </li><li> 7-8 ore più tardi, set-up co-culture seminando 250.000 mTECs (sia mTECs completi, o CD80 e CD80 lo sottoinsiemi hi), in un volume di 100 ml per pozzetto in cima al ponteggio fibroblasti. Contare le cellule utilizzando una camera di Neubauer (4 ° giorno). NOTA: In ogni momento le timo colture organotipiche 3D sono sommerse dai media a differenza OTC della pelle, che sono dotate di aria sollevato. </li><li> Dopo 24 ore di incubazione, di fornire le culture con medio (totale medio di cambio), come detto sopra (punto 2.3.2) ora containing ridotta quantità di Aprotinina, 250 unità / ml (Giorno 5). </li><li> Per valutare l&#39;attività proliferativa delle mTECs, aggiungere EdU (6,7 mM / ml, cioè, 10 mM / pozzetto) per OTCs per 4 ore prima della cessazione delle culture. Eseguire la colorazione di OTC crio-sezioni come descritto nel Kit EdU Imaging combinata con la co-colorazione di cheratina 14. Determinare gli indici proliferative mTECs, da una delle contando cellule K14 + EdU + in due sezioni di ogni esemplare la cultura o di flusso citometria (EdU citometria a flusso, Sezione 1.7 ma invece di Ly51-FITC utilizzano CDR1-PB 15 ad una diluizione 1: 100 e 2.3.6.4). </li><li> Dopo 4-7 giorni di co-coltura, terminare i farmaci da banco e il processo per l&#39;isolamento di RNA, crio-sezionamento o FACS analisi (giorno 8-11). <ol><li> Terminate le culture utilizzando una pinza, separando l&#39;equivalente scaffold / cutanea dal filtro dell&#39;inserto bene. </li><li> Per crio-sezionamento, incorporare l&#39;intero OTC in OCT e congelare in liquid vapori di azoto prima crio-sezionamento. Preparare 5-7 micron di spessore sezioni OTC utilizzando un criostato e conservare a -20 ° C fino al momento dell&#39;uso. Per immunoistochimica di OTC crio-sezione usare anti-cheratina 14 (AF64, uso a 1: 1.000 diluizione), e anti-vimentina (GP53, uso a 1: 100 diluizione) anticorpi. Eseguire la colorazione immunoistochimica indiretta utilizzando i rispettivi anticorpi secondari. </li><li> Per l&#39;isolamento di RNA, aggiungere 1 ml di denaturazione soluzione (contenente fenolo e guanidina tiocianato) in un tappo a vite di un tubo libero 2 ml RNasi. Tagliare l&#39;intera OTC in pezzi con un bisturi e aggiungere nella provetta contenente soluzione denaturazione. Meccanicamente sminuzzare i OTCs con strumento FastPrep due volte per 30 secondi a una velocità di 6,0, inserire tra il ghiaccio per 2 min (il campione può essere conservato a -80 ° C dopo questo punto). Se congelato a -80 ° C, scongelare su ghiaccio. Centrifugare le provette a 11,500-13,000 rpm per 10 minuti a 4 ° C. Trasferire il surnatante in una nuova provetta, incubare per 5 minuti a temperatura ambiente e fprotocolli di isolamento RNA susseguirsi utilizzando guanidinio estrazione tiocianato-fenolo-cloroformio acido come descritto dal produttore. </li><li> Per l&#39;analisi FACS, rimuovere l&#39;impalcatura e separare la membrana della fibrina / fibroblasti / gel MTEC. Finemente tagliare il gel con un bisturi e digerire in un tubo FACS per ~ 20 minuti o fino a quando completamente digerito con 2 ml di collagenasi / dispasi a 37 ° C in un bagno d&#39;acqua con agitazione magnetica. Agitare la soluzione enzimatica con una pipetta Pasteur volta ogni 5 min. Dopo completa digestione, filtrare la sospensione cellulare attraverso un filtro 70 micron; macchiare la sospensione singola cella utilizzando gli anticorpi come descritto in 1.7 e analizzare mediante citometria a flusso. </li></ol></li></ol></li></ol>

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Gli autori dichiarano assenza di conflitto finanziaria o commerciale di interesse.

Accanto RTOCs, l&#39;OTC 3D sono stati di gran lunga superiore in termini di differenziazione TEC e PGE manutenzione / induzione (Tabella 1) rispetto ad altri (i) &#39;culture 3D semplificati&#39; utilizzando - fibroblasti da solo senza il patibolo; (Ii) i sistemi 2D utilizzando - cellule fibroblasti / alimentatore co-coltura con TEC 10, (iii) le cellule 3T3-J2 cui cloni TEC sviluppano, ma PGE è perso, (iv) matrigel o (v) componenti ECM (dati non pubblicati). PGE è stato mantenuto fino a 7 ...

Timociti sviluppo costituiscono circa il 98% del timo, mentre il restante 2% è costituito da una varietà di cellule che compongono collettivamente stroma timico (cioè, cellule epiteliali, cellule dendritiche, macrofagi, cellule B, fibroblasti, cellule endoteliali). Le cellule epiteliali esterni corticali (CTEC) procurano immigrazione di cellule pro-T dal midollo osseo, induzione linea cellulare T in multipotenti cellule pre-T e selezione positiva di auto-MHC limitati timociti immaturi. Le cellule interne midollari timiche epiteliali (mTECs) sono coinvolti nella induzione della tolleranza di questi timociti con un alta affinità TCR per complessi auto-peptide / MHC da uno inducendo selezione negativa o loro deviazione nella linea cellulare T normativo. Nel contesto di induzione della tolleranza centrale, mTECs sono unici in quanto esprimono un ampio spettro di auto-antigeni tissutali-limitato (Tras) rispecchiando così il sé periferica. Questo fenomeno è chiamato espressione genica promiscua (PGE)1,2. La maggior parte degli studi in corso su questo tipo di cellule affascinante si basano su cellule isolate ex vivo, come i vari sistemi di coltura 2D a breve termine invariabilmente portato alla perdita di PGE e molecole regolatore chiave come MHC di classe II, FoxN1 e Aire entro i primi 2 giorni 3-6 . Rimase tuttavia poco chiaro, che particolari componenti e le caratteristiche del reticolo 3D intatto del timo mancavano in modelli 2D. La riaggregazione coltura dell&#39;organo timica (RTOC) è stato finora l&#39;unico sistema 3D che permette lo studio dello sviluppo delle cellule T, da una parte, e la biologia cellule stromali, d&#39;altra parte, in un microambiente timico intatta 7. Tuttavia, RTOCs hanno alcune limitazioni, cioè, essi contengono già una miscela complessa di cellule, richiederà il contributo delle cellule stromali fetali e sopportare un periodo di coltura massima di 5 a 10 giorni. La mancanza di riduzionista nei sistemi di coltura in vitro ha ostacolato lo studio didiversi aspetti di sviluppo delle cellule T e thymic organogenesi non ultima la regolazione molecolare di PGE e il suo rapporto con la biologia dello sviluppo di mTECs. A causa del primo relazionalità dell&#39;organizzazione strutturata delle cellule epiteliali della pelle e del timo, abbiamo optato per un sistema 3D cultura organotipica (OTC), che era stato sviluppato originariamente per emulare la differenziazione dei cheratinociti in vitro e creare un equivalente cutanea così. Il sistema OTC costituito da una matrice inerte scaffold sovrapposti con fibroblasti dermici che sono intrappolati in un gel di fibrina, su cui cheratinociti vengono seminate 8,9. Qui, abbiamo sostituito con cheratinociti mTECs purificati. Pur mantenendo le caratteristiche di base di questo modello, abbiamo ottimizzato alcuni parametri. Nel modello OTC adottato mTECs proliferato, ha subito la differenziazione terminale e mantenuto l&#39;identità MTEC e PGE, quindi mimando in vivo sviluppo mTECs <sup&gt; 10. Questa nota tecnica fornisce un protocollo dettagliato che permette la graduale messa a punto di OTC timo.

<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

Lo sviluppo delle cellule T Intra-timico richiede un reticolo tridimensionale complessa composta da varie cellule stromali, cioè cellule non-T. Timociti attraversano questo patibolo in un ordine temporale e spaziale altamente coordinato, mentre in sequenza passa punti di controllo obbligatori, cioè, l&#39;impegno linea cellulare T, seguita da generazione repertorio recettore delle cellule T e la selezione prima della loro esportazione nella periferia. I due principali tipi di cellule che formano residente questa impalcatura sono cellule epiteliali del timo corticali (CTEC) e midollari (mTECs). Una caratteristica fondamentale di mTECs è la cosiddetta espressione promiscua di numerosi antigeni tissutali limitato. Questi antigeni tissutali con restrizioni vengono presentati immaturi timociti direttamente o indirettamente da mTECs o cellule dendritiche del timo, causando rispettivamente in self-tolleranza. Adatto in vitro modelli che emulano i percorsi di sviluppo e le funzioni di CTEC e mTECs sono attualmente lacre. Questa mancanza di adeguati modelli sperimentali è per esempio ostacolato l&#39;analisi dell&#39;espressione genica promiscua, che è ancora poco conosciuta a livello cellulare e molecolare. Abbiamo adattato una organotipica modello di co-coltura 3D per la cultura ex vivo mTECs isolati. Questo modello è stato originariamente concepita per coltivare cheratinociti in modo tale da generare un equivalente pelle in vitro. Il modello 3D conservato caratteristiche funzionali chiave di MTEC biologia: (i) la proliferazione e la differenziazione terminale di CD80 ecco, Aire-negativo in CD80 hi, Aire-positivi mTECs, (ii) la risposta a RANKL, e (iii) espressione sostenuta di FoxN1, Aire e geni tessuto-limitato a CD80 mTECs hi.

Abbiamo adottato un organotipica co-coltura modello 3D (3D OTC) che era stato originariamente sviluppato per la cultura a lungo termine in vitro dei cheratinociti 9. MTECs MACS-arricchiti (vedi MACS arricchimento schema Figura 1) sono state seminate su un ponteggio composto da un gel di fibrina e fibroblasti intrappolati. I fibroblasti forniscono la matrice extracellulare essenziale (ECM) sostegno mTECs in vitro. MTECs sono state coltivate in OTC per 4-14 giorni, in presenza...

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.

3D organotipica Co-cultura Modello Sostenere midollare timica epiteliale proliferazione, differenziazione e promiscua genica

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 Views.  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. 

Video: 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.

Cultura e co-coltura di mouse ovaie e follicoli ovarici

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

Ricerca

Biologia dello sviluppo

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.

Previsione e Validazione del gene regolatore elementi attivato durante retinoico Acid Induced embrionali cellule staminali Differenziazione

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.

Adenovirale Terapia genica per diabetici cheratopatia: Effetti sulla guarigione delle ferite e Stem Cell Marker Expression in Human Organ cornee-coltura e le cellule epiteliali Limbal

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.

Temporale ordinazione di espressione Dynamic Data di espressione spaziale dettagliata Mappe

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.

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

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.

Protocollo di differenziazione cerebellare 3D semplificata con modifica 2D opzionale

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.

Analisi clonale di cellule embrionali staminali ematopoietiche precursori utilizzando indice della singola cella ordinamento combinato con cellule endoteliali nicchia co-cultura

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.

Proliferazione e differenziazione dei precursori mieloidi murino 32D/G-CSF-R cellule

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.

Riprogrammazione emogenica dei fibroblasti umani mediante l'espressione forzata dei fattori di trascrizione

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.

Un modello di espressione della chemiochina ectopica per testare il reclutamento macrofrofo 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 ...