Ce travail a été financé par une séquence PRN Frontiers in Genetics attribution du pilote, Juvenile Diabetes Research Foundation Grant et Grant 41-2009-775 12-126875 de Sygdom de Det Frie Forskningsråd / Sundhed. Les auteurs remercient le laboratoire Spagnoli pour accueillir le tournage vidéo.

Ce protocole a pour but de développer organites pancréatiques murines dérivées de E10.5 dissociées des cellules épithéliales pancréatiques. Le protocole exige l&#39;approbation éthique pour l&#39;expérimentation animale. 1. Dissection de Dorsale pancréatique Bud de E10.5 embryons de souris <ol><li> Moyen essentiel sacrifier souris expiré enceintes à jour embryonnaire (E) 10.5, ouvrez l&#39;abdomen avec une paire de ciseaux, enlever les deux cornes utérines et les placer dans une boîte de 10 cm de Pétri remplie avec du phosphate froid tampon salin (PBS) ou Dulbecco modifié (DMEM) conservé dans la glace. L&#39;expérience totale du sacrifice à l&#39;ensemencement des cellules est réalisé en 60 à 90 min pour prévenir les dommages cellulaires. </li><li> Séparer l&#39;utérus en segments d&#39;embryons individuels à l&#39;aide de petits ciseaux. Transférer un embryon dans une boîte de Petri de 35 mm avec du PBS froid et le visualiser sous un microscope à dissection avec l&#39;illumination d&#39;en haut. Avec des pinces à dissection enlever le muscle, caduque et jaune entourant sac et exposer l&#39;embryon. Placez l&#39;embryon de retour dans le milieu de DMEM froid. Les embryons peuvent être facilement transférées en utilisant un 3 ml transfert de plastique pipette / compte-gouttes. Isoler chaque embryon individuel d&#39;une manière similaire avant de poursuivre. </li><li> Placez un embryon dans une boîte de 35 mm propre Petri dans du PBS. <ol><li> Utilisez une pince fine (0,05 mm de largeur) pour enlever la patte antérieure. Insérer doucement la pince dans l&#39;ouverture pour détacher le tube digestif, de la région de la moelle épinière (figure 1). OPTION: Pour voir plus facilement l&#39;estomac, retirez la partie supérieure du corps de l&#39;embryon, dans le vif et la région de la queue en dessous de la tige de jaune. </li><li> Localiser l&#39;estomac, le foie et l&#39;intestin. Utilisation de la pince, d&#39;isoler le tractus gastro-intestinal entre l&#39;estomac et l&#39;intestin et le placer dans du DMEM froid sur de la glace (figure 1). Le bourgeon pancréatique dorsal est attaché le dos, postérieur à l&#39;estomac (Figure 1). </li><li> Isoler tous les tractus gastro-intestinal individu dans une similar manière avant de poursuivre. Si les embryons doivent être génotypés, recueillir la queue au moment de la dissection et de garder tous les tractus gastro-intestinal individu dans une bien différente dans une plaque de 24 puits avec du DMEM froid sur la glace. </li></ol></li><li> Placez un tractus gastro-intestinal dans une boîte de 35 mm propre Petri dans du PBS froid. Maintenant, utilisez un éclairage par le dessous dans le champ lumineux de visualiser et de disséquer le bourgeon pancréatique dorsal sous le microscope de dissection. Ces conditions permettent d&#39;optimiser la visualisation des volumes. En utilisant des aiguilles de tungstène affûtées par voie électrolytique ou 20 g des aiguilles de seringues, d&#39;isoler le bourgeon pancréatique avec aussi peu que possible mésenchyme autour de l&#39;épithélium (figure 1). <ol><li> Transférer l&#39;œuf isolé dans une boîte de Pétri contenant une solution de dispase froid (1,25 mg / ml) pendant 2-3 min. De ce point, le transfert peut être effectué le plus commodément par des capillaires en verre 50 ul de flamme tiré attachés à un tube flexible bouche contrôlée. Alternativement, mais avec plus de risk de perdre l&#39;œuf, utiliser une pipette 10 ul automatique (Pipetman) avec des pointes en plastique appropriées. </li><li> Effectuer pancréatique aspiration des bourgeons et l&#39;éjection sous contrôle microscopique. Mettez le bourgeon pancréatique retour dans PBS. Nettoyer en outre le bourgeon pancréatique isolé du mésenchyme avec les aiguilles et les aspiration douce à l&#39;aide du capillaire en verre (Figure 1). </li><li> Lorsque l&#39;ensemble du mésenchyme est enlevée, rincer le bourgeon pancréatique dans du PBS froid; transférer chaque bourgeon de DMEM froid dans des puits individuels (60 et mini-plateaux remplis de 10 ul de DMEM froid). Il est important de ne pas enlever le mésenchyme dans dispase, ce qui rend le tissu très collant. </li></ol></li></ol> 2. Placage et de la culture de cellules dispersées <ol><li> Transférer l&#39;épithélium disséqué à partir de tous les embryons avec un capillaire de verre à la flamme tiré dans des puits coniques de 60 puits mini-plateaux remplis avec 10 ul de PBS pour le rinçage. <ol><li> Transférer l&#39;œuf dans 10 ul de trypsine 0,05% unee laisser incuber à 37 ° C pendant 4 min. Inactiver la trypsine par le transfert de l&#39;œuf dans un puits avec 10 ul de DMEM + 10% de sérum foetal de veau (FCS). </li><li> Dissocier la suspension cellulaire par aspiration à travers un capillaire mince tiré avec un extracteur de pipette. Il est important d&#39;éviter les bulles à ce stade tout en aspiration et refoulement de dissocier les cellules. organites du pancréas commencent optimale de petits groupes de 5-15 cellules et donc dissociation partielle est recommandé (Figure 1). </li></ol></li><li> Réunir les cellules provenant de plusieurs embryons dans un tube Eppendorf afin de minimiser les différences dues au traitement individuel. Diluer la suspension de cellules dans le Matrigel refroidi dans un rapport 1:3. Aliquote de ce mélange sur une plaque de 96 puits, 8 pl / puits ou dans une plaque optimisé pour l&#39;imagerie (voir ci-dessous). </li><li> Incuber la plaque à 37 ° C pendant 5 min, ce qui permet d&#39;épaissir le Matrigel. Remplir les puits avec 70 pi de milieu de choix (organoïde ou une sphère, voir Tables figures 1 et 2) et laisser dans un environnement humidifié contenant 5% de CO2 et 95% d&#39;air à 37 ° C. </li><li> Remplacez le support tous les 4 e jour. Surveiller les colonies du pancréas augmente chaque jour et documenter le processus par imagerie. </li><li> Les petites molécules ou des protéines d&#39;intérêt peuvent être ajoutés au milieu à ce stade, pour des expériences d&#39;interférence, comme 28 indiqué précédemment. </li></ol> Tableau 1: moyen Organoid. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Nom de la matière </td><td> Stock Concentration </td><td> Concentration finale dans le milieu </td><td> Volume de stock </td></tr><tr><td> Pénicilline-streptomycine </td><td> 100% </td><td> 1% </td><td> 50 pl </td></tr><tr><td> Sérum remplacement KnockOut (supplément) </td><td> 100% </td><td> 10% </td><td> 500 pl </td></tr><tr><td> 2-mercaptoéthanol </td><td> 14,3 M </td&gt; <td> 0,1 mM </td><td> 1 pl </td></tr><tr><td> Phorbol myristate acétate (PMA) </td><td> 16 uM </td><td> 16 nM </td><td> 5 pl </td></tr><tr><td> Y-27632 (inhibiteur de ROCK) </td><td> 50 mM </td><td> 10 uM </td><td> 1 pl </td></tr><tr><td> EGF </td><td> 50 pg / ml </td><td> 25 ng / ml </td><td> 2,5 pl </td></tr><tr><td> Humaine recombinante R-1 Spondin </td><td> 250 pg / ml </td><td> 500 ng / ml </td><td> 10 pl </td></tr><tr><td> - Ou - </td><td></td><td></td><td></td></tr><tr><td> Recombinant de souris R-1 Spondin </td><td> 250 pg / ml </td><td> 500 ng / ml </td><td> 10 pl </td></tr><tr><td> Recombinant humain FGF1 (aFGF) </td><td> 100 pg / ml </td><td> 25 pg / ml </td><td> 1,25 pi </td></tr><tr><td> Héparine (Liquemin) </td><td> 2500 U / ml </td><td> 2,5 U / ml </td><td> 2 pl </td></tr><tr><td> RecomFGF10 humaine recombinante </td><td> 100 pg / ml </td><td> 100 ng / ml </td><td> 5 pl </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 pi </td></tr><tr><td> Total </td><td></td><td></td><td> 5000 pi </td></tr></tbody></table> Tableau 2: moyen Sphère. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Nom de la matière </td><td> Stock Concentration </td><td> Concentration finale dans le milieu </td><td> Volume de stock </td></tr><tr><td> Pénicilline-streptomycine </td><td> 100% </td><td> 1% </td><td> 50 pl </td></tr><tr><td> B27 x50 (supplément) </td><td> 100% </td><td> 10% </td><td> 100 pl </td></tr><tr><td> FGF2 recombinant humain (bFGF) </td><td> 100 pg / ml </td><td> 64 ng / ml </td><td> 3,2 pl </td></tr><tr><td> Y-27632 (inhibiteur de ROCK) </td><td> 50 mM </td><td> 10 uM </td><td> 1 pl </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845,8 ul </td></tr><tr><td> Total </td><td></td><td></td><td> 5000 pi </td></tr></tbody></table> 3. Imagerie de la progression du développement Organoid <ol><li> organoïdes Image soit quotidienne ou par faute de temps microscopie fluorescente utilisant un time-lapse microscope. Pour lapse imagerie en temps, utiliser un XY (Z) automatisé du microscope à fluorescence inversé. </li><li> De dépôt de petites gouttelettes de 3 ul dans des plaques à 4 puits ou des plaques à fond de verre remplies de 2-5 ml de milieu. Image avec un objectif 10x longue distance. Remarque: Les souris transgéniques exprimant des traceurs fluorescents peuvent être utilisés. Film 1 montre par exemple l&#39;expansion initiale de organoïdes de Pdx1-Ngn3-ER TM-IRES-NGFP + souris 4. La GFP nucléaire permet à l&#39;utilisateur de suivre les cellules sous forme d&#39;objets individuels, mais les mêmes principes peuvent être appliqués pour suivre les cellules présentant une fluorescence de membrane tellecomme mT / mG souris 29. </li><li> Lancer imagerie time-lapse 3 heures après l&#39;ensemencement des cellules pour éviter les dérives de discussion et configurer le logiciel d&#39;automatisation de contrôle pour prendre une photo / h pendant 3 jours ou plus à des positions définies manuellement. Pour chaque position, l&#39;acquisition d&#39;une interférence de contraste de l&#39;image différentielle (DIC) ainsi que le signal de la GFP, de rapports expression du marqueur fluorescent. </li></ol> 4. Recouvrement des organites pour histologie <ol><li> Placer la plaque de 96 puits sur la glace et retirez le support, le remplacer par PBS glacé. Cette dépolymérise partiellement Matrigel. </li><li> Aspirer doucement chaque individu organoïde, retirer le Matrigel entourant l&#39;aide d&#39;une pointe de 1000 pi afin de ne pas perturber l&#39;architecture globale. Transférer chaque organoïde à un bien avec le froid de la glace PBS. Conservez la plaque sur la glace. Fixation directe dans le Matrigel est également possible. </li><li> Fixer le organoïde pendant 15 min dans du paraformaldéhyde 4% (PFA), la cryoconservation en saccharose et l&#39;intégrer dans la gélatine. Traiter chaqueorganoïde pour cryosectioning et histologie comme décrit précédemment (Johansson et al., 2007) 4. </li></ol> 5. Recouvrement des organites pour la PCR et de biochimie <ol><li> Placer la plaque de 96 puits sur la glace et retirez le support. Ajouter 60 ul de RNAlater par puits afin de stabiliser et de protéger l&#39;ARN cellulaire. </li><li> Perturber le gel dans chaque puits mécaniquement par dépolymérisation partielle sur la glace. Soit récupérer organites individuels en utilisant une pointe de 1000 pi afin de ne pas perturber l&#39;architecture globale ou de récupérer la totalité de bien (avec Matrigel) en perturbant le gel mécaniquement avec une pointe de 200 pi. Utilisez une pointe de 1000 pi de transférer le contenu de puits dans un libre-non-collant le tube Eppendorf RNAse conservé dans la glace. </li><li> Laver les puits avec 60 pi RNAlater et ajouter le contenu restant dans le même tube Eppendorf. </li><li> Faites tourner les tubes pendant 5 min à 500-1000 g à 4 ° C. </li><li> Retirer le surnageant, ne laissant 20-30 pide RNAlater dans le tube en même temps que la pastille. Pour la biochimie, stocker les échantillons secs que possible. </li><li> Ne pas congeler les échantillons dans RNAlater immédiatement; conserver à 4 ° CO / N (pour permettre KNAlater de pénétrer en profondeur le tissu). Le tissu peut être conservé à -20 ° C pour une conservation à long terme et peut ensuite être traité pour la rupture des tissus et de l&#39;extraction de petites quantités d&#39;ARN. </li></ol>

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The authors have nothing to disclose.

La production à grande échelle de cellules bêta fonctionnelles in vitro est encore inefficace 1. Dans ce contexte difficile, les études de biologie du développement peuvent aider à déchiffrer les signaux exactes qui sont nécessaires à la différenciation des cellules bêta fonctionnelles. Ce protocole permet le maintien, l&#39;expansion et la différenciation des progéniteurs pancréatiques embryonnaires in vitro. Cela comprend la formation des cellules bêta productrices d&#39;ins...

culture d&#39;organes constitue un modèle utile qui comble le fossé entre le complexe mais très pertinente dans les enquêtes in vivo et la simulation pratique mais approximative de modèles de lignées cellulaires. Dans le cas du pancréas, il n&#39;y a pas de ligne parfaitement équivalente à progéniteurs pancréatiques bien qu&#39;il existe des lignées cellulaires transformées simulant des cellules endocrines et exocrines cellule. L&#39;adulte pancréas entier ne peut pas être cultivé; îlots endocrines isolées peuvent être maintenus pendant quelques semaines sans prolifération cellulaire et des tranches de tissu peuvent être conservées in vitro pendant 5 quelques heures. Culture de pancréas embryonnaire a été largement utilisé non seulement pour étudier son développement, mais aussi pour étudier les interactions épithéliales-mésenchymateuses 4,6,7, à l&#39;image des processus 8 ou interférer chimiquement avec eux 9. Deux méthodes de culture d&#39;organes sont principalement utilisés: la première consiste à cultiver des bourgeons pancréatiques sur la fibronectine plaques enduites de 2, ce qui est convenient à des fins d&#39;imagerie; la deuxième option est la culture des organes sur les filtres à l&#39;interface air-liquide qui préserve mieux 3,4 morphogenèse. Bien que très utiles, ces procédés conduisent à un certain degré d&#39;aplatissement; l&#39;expansion des progéniteurs est très limité par rapport au développement normal et à la population de départ est complexe comprenant tous les types de cellules pancréatiques et des cellules mésenchymateuses. La capacité de la culture et de développer des cellules primaires dispersées est précieux pour étudier les relations de lignage et de découvrir les propriétés intrinsèques des types de cellules isolées 10. Sugiyama et al. 11 pourrait maintenir les progéniteurs du pancréas et des progéniteurs endocrines qui ont conservé certains caractères fonctionnels pour 3-5 jours de culture sur des couches nourricières. Pancreatospheres, semblables à neurosphères 12 et mammospheres 13, ont été élargies de îlots adultes et les cellules canalaires bien que la nature des progéniteurs / cellules souchesqui génèrent ces sphères n&#39;est pas claire. De plus, en contraste avec le développement physiologique, les pancreatospheres contenaient certains neurones 14,15. Sphères ont été récemment produites à partir de progéniteurs pancréatiques embryonnaires 16,17 et régénération pancréas 18 avec une bonne extension des progéniteurs et la différenciation ultérieure, mais n&#39;ont pas réussi à récapituler la morphogenèse. Des modèles 3D à partir de cellules dispersées et souvent définis que l&#39;auto-organisent dans les organes miniaturisés ont récemment fleuri et simuler le développement ou adulte chiffre d&#39;affaires de plusieurs organes tels que l&#39;intestin 19,20, l&#39;estomac 21, le foie 22, la prostate 23 et la trachée 24. Dans certains cas, la morphogenèse et la différenciation de développement ont été récapitulé en 3D à partir de cellules ES, comme c&#39;est le cas de coupes optiques 25, de l&#39;intestin ou du cerveau 26 27. Ici, nous avons desCribe un moyen d&#39;étendre progéniteurs pancréatiques multipotentes dissociées dans un échafaudage 3D Matrigel où ils peuvent se différencier et s&#39;auto-organiser.

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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>
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			<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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		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
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		<tr>
			<td>&#160;- or -&#160;</td>
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			<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>
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		<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>
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			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
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		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
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			<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>
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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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		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
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			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
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		<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>
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		<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>
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		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
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		<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>
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		<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>
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		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
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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 dorsales progéniteurs pancréatiques dissociées et ensemencées en 3D Matrigel récapitulent le développement du pancréas. Progéniteurs peuvent être le plus facilement suivies avec les journalistes fluorescentes. Dans notre cas nous avons utilisé une souris transgénique qui exprime une protéine GFP nucléaire contrôlée par Pdx1 promoteur (Pdx1-Ngn3-ER TM-NGFP) (Film 1) en l&#39;absence de tamoxifène et donc sans activation Neurog3 4 (figure 2). <...

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

Le procédé de culture en trois dimensions décrite dans ce protocole récapitule le développement du pancréas de progéniteurs embryonnaires dispersées de pancréas de souris, y compris leur expansion substantielle, la morphogenèse et la différenciation dans un organe ramifié. Ce procédé se prête à l&#39;imagerie, la perturbation fonctionnelle et la manipulation de la niche.

In Vitro pancréas organogenèse du dispersées progéniteurs embryonnaires de souris

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

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JoVE Journal

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K vues.  Swiss Institute for Experimental Cancer Research.  Le procédé de culture en trois dimensions décrite dans ce protocole récapitule le développement du pancréas de progéniteurs embryonnaires dispersées de pancréas de souris, y compris leur expansion substantielle, la morphogenèse et la différenciation dans un organe ramifié. Ce procédé se prête à l'imagerie, la perturbation fonctionnelle et la manipulation de la niche. 

Vidéo: 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.

Différenciation in vitro de souris embryonnaires souches (MES) Cellules utilisant la méthode de goutte suspendue

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 ...

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Biologie

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.

Enregistrements aspiration simple cellule à partir de cônes photorécepteurs de la souris

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.

L&#39;imagerie in vivo et des traitements thérapeutiques dans un modèle de souris orthotopique de cancer des ovaires

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.

Purification des progéniteurs du muscle squelettique

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 système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

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.

Génération de souris dérivées de cellules souches pluripotentes induites

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.

Cellule étiquetage et l&#39;injection dans le développement embryonnaires coeurs de souris

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.

Isolement de l&#39;ARN de souris Pancréas: un tissu de Ribonuclease riche

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.

Protein Isolation du développement embryonnaire Souris Coeur Région Valve

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.

Procédure pour évaluer l&#39;efficacité de floculants pour l&#39;élimination des particules dispersées dans des extraits de plantes

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 ...