Questo lavoro è stato finanziato in sequenza da un PRN Frontiere della genetica premio pilota, Juvenile Diabetes Research Foundation Grant e Grant 41-2009-775 12-126875 da Det Frie forskningsråd / Sundhed og Sygdom. Gli autori ringraziano il laboratorio Spagnoli per ospitare le riprese video.

Questo protocollo mira a far crescere organoidi pancreatiche derivate da E10.5 murino dissociate cellule pancreatiche epiteliali. Il protocollo richiede l&#39;approvazione etica per la sperimentazione animale. 1. Dissezione della gemma dorsale del pancreas da E10.5 embrioni di topo <ol><li> Sacrifica il topo gravide temporizzata a giorno embrionale (E) 10.5, aprire l&#39;addome con un paio di forbici, rimuovere le due corna uterine e metterli in un piatto di 10 centimetri di Petri riempita con fosfato freddo tampone salino (PBS) o Dulbecco modificato mezzo essenziale (DMEM) tenuta in ghiaccio. L&#39;esperimento totale dal sacrificio di semina delle cellule avviene in 60-90 min per prevenire danni cellulari. </li><li> Separare l&#39;utero in singoli segmenti di embrioni utilizzando piccole forbici. Trasferire un embrione di un piatto 35 millimetri Petri con PBS freddo e visualizzare sotto un microscopio da dissezione con illuminazione dall&#39;alto. Con una pinza dissezione rimuovere il muscolo, decidua e tuorlo circostante sac ed esporre l&#39;embrione. Posizionare l&#39;embrione di nuovo in mezzo DMEM freddo. Gli embrioni possono essere facilmente trasferiti utilizzando 3 ml di trasferimento di plastica pipetta / contagocce. Isolare ogni singolo embrione in modo simile prima di continuare. </li><li> Inserire un embrione in un 35 millimetri piatto pulito Petri in PBS. <ol><li> Utilizzare pinze sottili (0,05 mm di larghezza) per rimuovere la zampa anteriore. Inserire delicatamente la pinza nell&#39;apertura per staccare il tubo digerente dalla regione del midollo spinale (Figura 1). OPTIONAL: Per vedere più comodamente lo stomaco, rimuovere la parte superiore del corpo dell&#39;embrione, fino al cuore e la regione di coda sotto il gambo tuorlo. </li><li> Individuare lo stomaco, il fegato e l&#39;intestino. Utilizzando le pinze, isolare il tratto gastrointestinale dallo stomaco all&#39;intestino e trasferirla in DMEM freddo su ghiaccio (Figura 1). Il germoglio pancreatico dorsale è attaccato dorsalmente, posteriormente allo stomaco (Figura 1). </li><li> Isolare ogni individuo tratto gastrointestinale in un Similar modo prima di continuare. Se devono essere genotipizzati gli embrioni, raccogliere la coda al momento della dissezione e mantenere ogni singolo tratto gastrointestinale in un ben diverso in una piastra da 24 pozzetti con DMEM freddo sul ghiaccio. </li></ol></li><li> Posizionare un tratto gastrointestinale in un 35 millimetri piatto pulito Petri in PBS freddo. Ora usate l&#39;illuminazione dal basso in campo chiaro di visualizzare e analizzare il bocciolo pancreatico dorsale sotto il microscopio di dissezione. Queste condizioni di ottimizzare la visualizzazione dei volumi. Usando aghi di tungsteno elettroliticamente-affilati o 20 aghi da siringa G, isolare la gemma pancreatica con il minor mesenchima possibile intorno l&#39;epitelio (Figura 1). <ol><li> Trasferire il germoglio isolato in una capsula petri contenente una soluzione dispasi fredda (1,25 mg / ml) per 2-3 min. Da questo punto, trasferimento può più convenientemente essere fatto con-tirato fiamma 50 capillari di vetro microlitri collegati ad un tubo flessibile bocca controllato. In alternativa, ma con più risk di perdere la gemma, utilizzare un 10 microlitri pipetta automatica (Pipetman) con appropriati consigli di plastica. </li><li> Eseguire pancreatica aspirazione nascere ed espulsione sotto controllo microscopico. Mettere la gemma pancreatica nel PBS. Ulteriori pulire la gemma pancreatica isolato dal mesenchima con gli aghi e aspirando delicatamente utilizzando la capillare di vetro (Figura 1). </li><li> Quando l&#39;intero mesenchima viene rimosso, sciacquare la gemma pancreatica in PBS freddo; trasferire ogni germoglio di DMEM freddo nei singoli pozzetti (60 pozzetti mini-vaschette riempite con 10 ml di DMEM freddo). È importante non rimuovere mesenchima in dispasi, che rende il tessuto molto appiccicoso. </li></ol></li></ol> 2. Placcatura e la coltura di cellule dislocati <ol><li> Trasferire gli epiteli sezionato da tutti gli embrioni con un bicchiere capillare tirato fiamma nei pozzetti conici di 60 pozzetti mini-vassoi riempiti con 10 microlitri di PBS per il risciacquo. <ol><li> Trasferire il germoglio in 10 ml di tripsina 0,05% and lasciate incubare a 37 ° C per 4 min. Inattivare la tripsina trasferendo il germoglio in un pozzo con 10 microlitri DMEM + 10% di siero fetale bovino (FCS). </li><li> Dissociare la sospensione cellulare per aspirazione attraverso una capillare sottile tirato con un estrattore pipetta. È importante evitare bolle in questa fase mentre pipettando su e giù per dissociare le cellule. Organoidi pancreas in modo ottimale a partire da piccoli gruppi di 5-15 cellule e quindi la dissociazione parziale è raccomandata (Figura 1). </li></ol></li><li> Si riuniscono le cellule da diversi embrioni in una provetta Eppendorf per minimizzare le differenze dovute al trattamento individuale. Diluire la sospensione cellulare in refrigerate Matrigel in un rapporto 1:3. Aliquotare questa miscela in una piastra a 96 pozzetti, 8 microlitri / pozzetto in una piastra o ottimizzato per l&#39;imaging (vedi sotto). </li><li> Incubare la piastra a 37 ° C per 5 minuti, consentendo al Matrigel per addensare. Riempire i pozzetti con 70 ml di mezzo di sua scelta (organoide o sfera, vedi Tables 1 e 2) e lasciare in un ambiente umidificata contenente il 5% di CO 2 e il 95% aria a 37 ° C. </li><li> Sostituire il mezzo ogni 4 ° giorno. Monitorare le crescenti colonie pancreatiche quotidiana e documentare il processo di imaging. </li><li> Piccole molecole o proteine ​​di interesse possono essere aggiunti al mezzo in questa fase per esperimenti di interferenza, come riportato in precedenza 28. </li></ol> Tabella 1: media organoide. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Nome del materiale </td><td> Archivio Concentrazione </td><td> Concentrazione nel mezzo finale </td><td> Volume di magazzino </td></tr><tr><td> Penicillina-Streptomicina </td><td> 100% </td><td> 1% </td><td> 50 microlitri </td></tr><tr><td> Sostituzione Serum KnockOut (supplemento) </td><td> 100% </td><td> 10% </td><td> 500 microlitri </td></tr><tr><td> 2-mercaptoetanolo </td><td> 14.3 M </td&gt; <td> 0,1 mm </td><td> 1 ml </td></tr><tr><td> Phorbol Myristate acetato (PMA) </td><td> 16 micron </td><td> 16 Nm </td><td> 5 microlitri </td></tr><tr><td> Y-27632 (inibitore ROCK) </td><td> 50 mM </td><td> 10 pM </td><td> 1 ml </td></tr><tr><td> EGF </td><td> 50 mg / ml </td><td> 25 ng / ml </td><td> 2,5 microlitri </td></tr><tr><td> Ricombinante umana R-spondina 1 </td><td> 250 mg / ml </td><td> 500 ng / ml </td><td> 10 microlitri </td></tr><tr><td> - O - </td><td></td><td></td><td></td></tr><tr><td> Ricombinante mouse R-spondina 1 </td><td> 250 mg / ml </td><td> 500 ng / ml </td><td> 10 microlitri </td></tr><tr><td> FGF1 ricombinante umano (aFGF) </td><td> 100 pg / ml </td><td> 25 mcg / ml </td><td> 1.25 microlitri </td></tr><tr><td> Eparina (Liquemin) </td><td> 2500 U / ml </td><td> 2,5 U / ml </td><td> 2 microlitri </td></tr><tr><td> Recombinant FGF10 umano </td><td> 100 pg / ml </td><td> 100 ng / ml </td><td> 5 microlitri </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 microlitri </td></tr><tr><td> Totale </td><td></td><td></td><td> 5.000 pl </td></tr></tbody></table> Tabella 2: Sphere medie. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Nome del materiale </td><td> Archivio Concentrazione </td><td> Concentrazione nel mezzo finale </td><td> Volume di magazzino </td></tr><tr><td> Penicillina-Streptomicina </td><td> 100% </td><td> 1% </td><td> 50 microlitri </td></tr><tr><td> B27 x50 (supplemento) </td><td> 100% </td><td> 10% </td><td> 100 microlitri </td></tr><tr><td> FGF2 ricombinante umano (bFGF) </td><td> 100 pg / ml </td><td> 64 ng / ml </td><td> 3.2 microlitri </td></tr><tr><td> Y-27632 (inibitore ROCK) </td><td> 50 mM </td><td> 10 pM </td><td> 1 ml </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4.845,8 ml </td></tr><tr><td> Totale </td><td></td><td></td><td> 5000 microlitri </td></tr></tbody></table> 3. Imaging della progressione dello Sviluppo organoide <ol><li> Organoidi fotografia sia giornaliero o mediante microscopia time lapse utilizzando un microscopio a fluorescenza time-lapse. Per l&#39;imaging lasso di tempo, utilizzare un XY (Z) microscopio a fluorescenza invertito automatizzato. </li><li> Deposito piccole goccioline di 3 ml in piastre 4 pozzetti o piastre con fondo di vetro riempiti con 2-5 ml di media. Immagine con un obiettivo 10x lunga distanza. Nota: I topi transgenici che esprimono traccianti fluorescenti possono essere utilizzati. Movie 1 mostra per esempio l&#39;espansione iniziale di organoidi da Pdx1-Ngn3-ER TM-IRES-nGFP + mouse 4. La GFP nucleare consente all&#39;utente di monitorare le cellule come singoli oggetti ma principi simili possono essere applicati per monitorare le cellule con membrana fluorescenza talecome mT / mG topi 29. </li><li> Inizia time-lapse imaging 3 ore dopo la semina delle cellule per evitare derive di messa a fuoco e impostare l&#39;automazione di controllo software di prendere 1 foto / h per 3 o più giorni in posizioni definite manualmente. Per ogni posizione, acquisire un contrasto interferenziale immagine (DIC), nonché il segnale GFP, notifica espressione marcatore fluorescente. </li></ol> 4. Recupero di organoidi di Istologia <ol><li> Posizionare la piastra a 96 pozzetti su ghiaccio e rimuovere il mezzo, sostituendolo con ghiaccio PBS freddo. Questo depolimerizza parzialmente Matrigel. </li><li> Aspirare delicatamente ogni singolo organoide, rimuovendo il Matrigel circostante con una punta di 1.000 ml, al fine di non perturbare l&#39;architettura complessiva. Trasferire ciascun organoide a un pozzo con ghiaccio freddo PBS. Mantenere la piastra su ghiaccio. Fissaggio diretto in Matrigel è anche possibile. </li><li> Fissare la organoide per 15 minuti a 4% paraformaldeide (PFA), criopreservare in saccarosio e incorporarlo in gelatina. Elaborare ogniorganoide per criosezionamento e istologia come precedentemente descritto (Johansson et al., 2007) 4. </li></ol> 5. Recupero di organoidi per la PCR e Biochimica <ol><li> Posizionare la piastra a 96 pozzetti su ghiaccio e rimuovere il supporto. Aggiungere 60 ml di RNAlater per pozzetto per stabilizzare e proteggere RNA cellulare. </li><li> Interrompere il gel in ogni pozzetto meccanicamente da parte depolimerizzante su ghiaccio. O ripristinare singoli organoidi con una punta di 1.000 ml, al fine di non perturbare l&#39;architettura generale o recuperare l&#39;intero bene (con Matrigel) interrompendo il gel meccanicamente con una punta di 200 microlitri. Utilizzare una punta di 1.000 microlitri di trasferire il contenuto e in un libero non appiccicosa provetta Eppendorf RNAsi tenuto in ghiaccio. </li><li> Lavare i pozzetti con 60 microlitri RNAlater e aggiungere il resto del contenuto alla stessa provetta Eppendorf. </li><li> Centrifugare le provette per 5 min a 500-1000 xga 4 ° C. </li><li> Rimuovere il surnatante, lasciando solo il 20-30 microlitridi RNAlater nel tubo insieme al pellet. Per biochimica, conservare i campioni più asciutto possibile. </li><li> Non congelare i campioni in RNAlater immediatamente; conservare a 4 ° CO / N (per consentire RNAlater di penetrare a fondo il tessuto). Il tessuto può essere conservato a -20 ° C per una conservazione a lungo termine e può essere successivamente trattati per frammentazione del tessuto e l&#39;estrazione di piccole quantità di RNA. </li></ol>

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The authors have nothing to disclose.

La produzione su larga scala delle cellule beta funzionali in vitro è ancora inefficace 1. In questo contesto impegnativo, studi di biologia dello sviluppo possono aiutare decifrare i segnali esatti che sono necessari per la differenziazione delle cellule beta funzionali. Questo protocollo consente per la manutenzione, l&#39;espansione e la differenziazione di progenitori pancreatici embrionali in vitro. Ciò include la formazione di cellule beta produttrici di insulina che non co-express a...

Cultura Organ fornisce un modello utile che colma il divario tra il complesso, ma di grande rilevanza nelle indagini vivo e conveniente, ma approssimativa simulazione di modelli di linee cellulari. Nel caso del pancreas, non vi è alcuna linea cellulare perfettamente equivalente a progenitori pancreas anche se ci si trasformano linee cellulari che simulano cellule endocrine ed esocrine. L&#39;intero pancreas adulto non può essere coltivato; isolato isolotti endocrini possono essere mantenuti per poche settimane senza proliferazione cellulare e fette di tessuto possono essere tenuti in vitro per alcune ore 5. Cultura pancreas embrionale è stato ampiamente utilizzato non solo per studiare il suo sviluppo, ma anche per indagare interazioni epitelio-mesenchimale 4,6,7, all&#39;immagine processi 8 o interferire chimicamente con essi 9. Due organi metodi di coltura sono utilizzati principalmente: la prima consiste nella coltura gemme pancreatiche su piastre rivestite di fibronectina 2, che è conveutile per gli scopi di imaging; la seconda opzione è quella di cultura organi sui filtri all&#39;interfaccia aria-liquido 3,4 che meglio conserva morfogenesi. Sebbene molto utile, questi metodi portano ad un certo grado di appiattimento; l&#39;espansione di progenitori è molto limitato rispetto al normale sviluppo e la popolazione di partenza è complesso comprendente tutti i tipi di cellule pancreatiche e cellule mesenchimali. La capacità di cultura ed espandere cellule primarie disperse è prezioso per studiare le relazioni lignaggio e scoprire le proprietà intrinseche dei tipi di cellule isolate 10. Sugiyama et al 11. Potrebbe mantenere progenitori pancreas e progenitori endocrini che hanno conservato alcuni caratteri funzionali per 3-5 giorni di coltura su livelli di alimentazione. Pancreatospheres, simile a neurospheres 12 e mammospheres 13, sono stati espansi da isolotti adulte e cellule duttali sebbene la natura dei progenitori / cellule staminaliche generano queste sfere non è chiaro. Inoltre, in contrasto con sviluppo fisiologico, i pancreatospheres contenevano alcuni neuroni 14,15. Spheres sono stati recentemente realizzati da progenitori pancreas embrionale 16,17 e rigenerante pancreas 18 con buona espansione progenitrici e la successiva differenziazione, ma non è riuscito a riepilogare morfogenesi. Modelli 3D da cellule disperse e spesso definiti che l&#39;auto-organizzano in organi miniaturizzati hanno recentemente fiorito e simulare lo sviluppo o adulto fatturato di più organi quali l&#39;intestino 19,20, 21 stomaco, il fegato 22, la prostata 23 e la trachea 24. In alcuni casi, la morfogenesi di sviluppo e differenziazione sono stati riassunti in 3D da cellule ES, come è il caso di tazze ottiche 25, 26 o intestino cerebrali 27. Qui, desCRIBE un metodo per espandere dissociate progenitori pancreatici multipotenti in un ponteggio 3D Matrigel dove possono differenziarsi e di auto-organizzarsi.

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			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
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		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
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			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
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			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
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			<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>
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			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
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			<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>
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		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
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			<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>
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</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.

E10.5 dorsali progenitori pancreatici dissociate e seminate in 3D Matrigel ricapitolano sviluppo del pancreas. Progenitori possono essere più facilmente seguiti con i giornalisti fluorescenti. Nel nostro caso abbiamo utilizzato un topo transgenico che esprime una proteina GFP nucleare controllata dal promotore Pdx1 (Pdx1-Ngn3-ER TM-nGFP) (film 1) in assenza di tamoxifene e quindi senza attivare NEUROG3 4 (Figura 2). Con il mezzo organoide, una...

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

Il metodo di coltura tridimensionale descritto in questo protocollo ricapitola sviluppo pancreas dispersi da progenitori topo pancreas embrionali, compresa la loro notevole espansione, differenziazione e morfogenesi in un organo ramificato. Questo metodo è suscettibile di immagini, interferenze funzionali e manipolazione della nicchia.

In Vitro Pancreas Organogenesis da dislocati embrionali di topo Progenitori

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 Views.  Swiss Institute for Experimental Cancer Research.  Il metodo di coltura tridimensionale descritto in questo protocollo ricapitola sviluppo pancreas dispersi da progenitori topo pancreas embrionali, compresa la loro notevole espansione, differenziazione e morfogenesi in un organo ramificato. Questo metodo è suscettibile di immagini, interferenze funzionali e manipolazione della nicchia. 

Video: 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.

La differenziazione in vitro di staminali embrionali di topo (MES) Cellule con il metodo goccia

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 ...

Ricerca

Biologia

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.

Unicellulari registrazioni di aspirazione da Fotorecettori Cone mouse

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.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

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.

Purificazione di progenitori dal muscolo scheletrico

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.

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

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.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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.

Etichettatura cellulare e iniezione nello sviluppo embrionali Cuori mouse

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.

Isolamento dell&#39;RNA da mouse Pancreas: Un Tessuto ricco Ribonucleasi

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.

Proteine ​​Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

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

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Procedura per valutare l&#39;efficienza di flocculanti per la rimozione di particelle disperse di estratti di piante

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...