Nous remercions U. Ganapati pour la création et l&#39;expérimentation du mStrawberry OP9 lignée cellulaire. De plus, nous remercions H. Shafifor son aide à la co-culture. HP est soutenue par une bourse de l&#39;Institut National Heart, Lung, and Blood (F31HL087714) (HP). Le contenu de ce travail est la seule responsabilité de l&#39;auteur et ne représentent pas nécessairement les vues officielles de l&#39;Institut National Heart, Lung, and Blood ou les National Institutes of Health (NIH). Le débit UCLA cytométrie Core Facility est soutenu par le NIH (CA-16 042 et AI-28697), le Jonsson Cancer Center, de l&#39;UCLA AIDS Institute et de l&#39;UCLA School of Medicine.

1. Préparation des cellules ES <ol><li> Les cellules ES culture selon le protocole standard pour votre lignée cellulaire donnée. Assurez-vous de préparer des cellules ES assez pour plaque sur les cellules OP9 mStrawberry pour la Journée 0 (cellules ES seront plaquées à 1x10 ^ 3 cellules par cm 2 au jour 0) </li></ol> 2. Préparation des cellules OP9 <ol><li> Avant croissante mStrawberry OP9 cellules, préparer le milieu de croissance OP9 OP-9 milieu de culture cellulaire <table border="1"><tbody><tr><td></td><td> Stock </td><td> Conc final. </td><td> Volume (ml) </td></tr><tr><td> AMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS (OP-9 testés) </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-glutamine </td><td> 100X (200mm) </td><td> 2mM </td><td> 0.5 </td></tr><tr><td></td><td></td><td></td><td> 50mL </td></tr></tbody></table> Conserver à 4 ° C et utiliser dans une semaine de préparation </li><li> Afin de maintenir mStrawberry OP9 cellules, les médias doivent être changés tous les 2 jours et les cellules doivent être repiquées avant d&#39;atteindre les cellules de confluence (subculture à 70-90% de confluence). cultures de cellules OP9 mStrawberry sont jamais grandi au delà de 90% de confluence comme cela se traduira dans leur différenciation en adipocytes. Voir Figure 3. <ol class="lalpha"><li> Repiquer les cellules par des cellules à laver avec du PBS 2X (ou 1x avec PBS 1x + 0,1% à la trypsine-EDTA) </li><li> Ajouter préchauffé 0,1% de trypsine-EDTA à 37 ° C pendant 5 minutes (ou jusqu&#39;à ce que les cellules se détacher) </li><li> Lorsque les cellules se sont détachées, ajouter moyennes OP-9 de la croissance. Transfert des cellules dans un tube de 50 ml conique. </li><li> Pellet cellules à 300 xg pendant 5 minutes à température ambiante. Rejeter le surnageant. </li><li> Resuspendre les cellules dans l&#39;eau douce mStrawberry OP9 milieux de croissance </li><li> Cellules de la plaque à une densité minimale de 10 4 cellules / cm 2 </li><li> Cultivez mStrawberry OP9 cellules à confluence commençant 3 jours avant le jour nécessaire pour les cellules ES co-culture (jours 0, 5, ou Jour8 / 9 de l&#39;expérience) </li></ol></li></ol> 3. ES / mStrawberry OP9 co-culture <ol><li> Préparez le support de différenciation co-culture <table border="1"><tbody><tr><td></td><td> Stock </td><td> Finale </td><td> Volume (ml) </td></tr><tr><td> AMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-glutamine </td><td> 200 mM </td><td> 2mM </td><td> 0.5 </td></tr><tr><td></td><td></td><td></td><td> 50mL </td></tr></tbody></table></li><li> mStrawberry OP9 cellules (Jour -3) <ol class="lalpha"><li> TOUJOURS Subculture OP9 cellules -3 jours, alors que vous avez couches confluentes de cellules OP9 mStrawberry pour 0 Jour de la co-culture. Voir la figure 4b. Plaque mStrawberry OP9 cellules à 1x10 4 / cm 2 pour confluence au jour 0. Soyez sûr de sous-culture des cellules supplémentaires pour Jour 5 et Jour 8 / 9 pour la poursuite de la co-culture. </li></ol></li><li class="jove_content"> ES / mStrawberry OP9 co-culture Set-Up (jour 0) <ol class="lalpha"><li class="jove_content"> Laver les cellules ES 2x avec du PBS </li><li> Trypsiniser cellules ES avec préchauffé 0,25% de trypsine-EDTA, incuber pendant 5min à 37 ° C </li><li> Ajouter le média de différenciation co-culture de cellules </li><li> Pellet cellules ES à 400xg, 5-7min, RT. Rejeter le surnageant </li><li> Resuspendre les cellules dans les médias de différenciation co-culture </li><li> Retirez le support du mStrawberry OP9 cellules </li><li> Ajouter le média de différenciation co-culture à mStrawberry flacons OP9 </li><li> Plaque 1x10 cellules ES 3 / cm 2 à flacon de mStrawberry OP9 cellules. Incuber les cellules à 37 ° C, 5% de CO 2 avec l&#39;humidité. </li></ol></li><li> mStrawberry OP9 cellules (Jour 2) <ol class="lalpha"><li> OP9 mStrawberry Subculture de sorte que vous avez des couches confluentes de cellules pour mStrawberry OP9 Jour 5 de la co-culture. Plaque mStrawberry OP9 cellules à 1x10 4 / cm 2 pour confluence au Jour 5 (comme dans l&#39;étape 3.2). Soyez sûr de sous-culture des cellules supplémentaires pour la Journée 8 / 9, si nécessaire (comme dans l&#39;étape 2.2). </li></ol></li><li> ES / mStrawberry OP9 co-culture (Jour 3) <ol class="lalpha"><li> Retirez délicatement moyennes à partir de co-culture avec des produits frais et de remplacer milieu de différenciation co-culture </li></ol></li><li> ES / mStrawberry OP9 co-culture (Jour 5) <ol class="lalpha"><li> Se laver soigneusement les co-cultures 2x avec du PBS </li><li> Trypsiniser cellules avec préchauffé 0,25% de trypsine-EDTA </li><li> Laver les cellules hors flacon avec les médias de différenciation co-culture </li><li> Cellules Pellet au 400xg, 7min, RT. Rejeter le surnageant </li><li>Ajouter le média de différenciation co-culture de cellules. Utiliser une aiguille de calibre 21 bluntended pour resuspendre les cellules et d&#39;assurer la suspension seule cellule </li><li> Compter les cellules en utilisant un hématimètre. Devrait avoir 100 à 150 fois plus en nombre de cellules ES du jour 0. </li><li> Réensemencez 6.48x10 4 / cm 2 - 7.76x10 4 / cm 2 de co-culture de cellules sur une nouvelle couche de confluence, les cellules OP9 mStrawberry </li><li> Jour 5 ESSAIS-restant co-culture des cellules peuvent être utilisées pour l&#39;analyse de cytométrie en flux, cytopreps (30K cellules), des dosages à puce, etc </li></ol></li><li> ES / mStrawberry OP9 co-culture (Jour 8 / 9 et la Journée 12) <ol class="lalpha"><li> Retirez le support de différenciation du ballon et sauver. Lavez monocouche 1x avec du PBS pour éliminer les cellules hématopoïétiques en suspension. Ne jetez pas sur les cellules hématopoïétiques. </li><li> Trypsiniser cellules adhérentes avec préchauffé 0,25% de trypsine-EDTA </li><li> Laver les cellules hors flacon avec les médias de différenciation ES et ajouter à cellules hématopoïétiques trouve en suspension = </li><li> Cellules Pellet au 400xg, 7min, RT. Rejeter le surnageant </li><li> Ajouter une différenciation co-culture des médias ou du PBS aux cellules. Utiliser une aiguille de calibre 21 bluntended pour resuspendre les cellules et d&#39;assurer pour les cellules de cellule unique Comte de suspension à l&#39;aide d&#39;un hémocytomètre. </li><li> Co-culture peut être poursuivi pendant 14 jours afin d&#39;évaluer plus mature progéniteurs hématopoïétiques par des re-semis de co-culture de cellules sur une nouvelle couche de confluence, les cellules OP9 mStrawberry au Jour 8 ou 9 puis à nouveau le jour 12. Réensemencez nombre de cellules optimisé basé sur la récupération des cellules nécessaires pour le jour de dosage. </li></ol></li><li> Évaluation des ES dérivées descendance (Jour 1-14). ES dérivées de la descendance peut être évaluée sur n&#39;importe quel jour de la co-culture par un écoulement tri mStrawberry cellules négatives (ES dérivées de descendance). <ol class="lalpha"><li> Retirez le support de différenciation du ballon et sauver. Lavez monocouche 1x avec du PBS pour éliminer les cellules hématopoïétiques en suspension. Ne jetez pas sur les cellules hématopoïétiques. </li><li> Trypsiniser cellules adhérentes avec préchauffé 0,25% de trypsine-EDTA </li><li> Laver les cellules hors flacon avec les médias de différenciation co-culture et l&#39;ajouter aux cellules hématopoïétiques trouve en suspension </li><li> Cellules Pellet au 400xg, 7min, RT. Rejeter le surnageant </li><li> Resuspendre les cellules avec 21 aiguille de calibre bluntended et compter </li><li> Descendances ES dérivées (mStrawberry cellules négatives) peuvent être triés du flux positifs mStrawberry OP9 cellules. Descendances ES dérivées peuvent alors être caractérisé par une analyse de cytométrie en flux, cytopreps, des dosages à puce, etc </li></ol></li></ol> 4. Les résultats représentatifs: <img src="/files/ftp_upload/2559/2559fig1.jpg" alt="Figure 1" /> Figure 1. ES / mStrawberry OP9 co-culture du système. Notre laboratoire utilise une étude in vitro de cellules ES de co-culture du système au modèle de développement hématopoïétique. Dans ce système de co-culture, les cellules ES de différencier, dans hémangioblastes au Jour 5, lorsqu&#39;il est placé sur une couche confluente de cellules mStrawberry OP9. Comme le produit de co-culture, précurseurs hématopoïétiques sont présents au Jour 8, tandis terminale différenciée lignées hématopoïétiques apparaissent au jour 14. Les flèches indiquent jours de différenciation lors du repiquage ES dérivées de la descendance sur de nouvelles couches confluentes de cellules dans mStrawberry OP9 nécessaire. <img src="/files/ftp_upload/2559/2559fig2.jpg" alt="Figure 2" /> Figure 2. Une bonne différenciation des cellules ES. Les cellules ES sont ensemencées sur une couche confluente de mStrawberry OP9 au jour 0. ES dérivées de la descendance commence à devenir visible au jour 3 de la différenciation, avec l&#39;ES-dérivée progéniture ressemble à un amas de cellules avec une frontière définie ou début pour former un motif verticille. Hémangioblastes, qui sont le précurseur commun mésodermiques pour les lignées endothéliales et hématopoïétiques, doit apparaître comme entassés verticilles de cellules au Jour 5. Pour la co-culture de plus longues périodes de cinq jours, ES dérivées de la descendance doit apparaître comme des amas de cellules hématopoïétiques. Deux à quatre pôles unicellulaires, au Jour 6 / 7, doit apparaître et se développer en des amas de cellules plus grandes par jour 8 / 9 de la co-culture. Descendances ES Autres dérivés sont également trouvés étroitement liée à la couche de cellules stromales adhérentes. <img src="/files/ftp_upload/2559/2559fig3.jpg" alt="Figure 3" /> Figure 3. Échec de différenciation des cellules ES. Les résultats de cette co-culture reflètent mauvaise différenciation des cellules ES. Notez comment la descendance ES dérivées n&#39;apparaissent pas dans un schéma verticille au Jour 5. L&#39;absence de cellules dans un modèle verticille indique la formation hémangioblaste incorrecte. Si hémangioblaste ne se développe pas correctement, puis a continué de co-culture se traduira dans la différenciation hématopoïétique lignée rabougris. Co-cultures avec la formation hémangioblaste incorrecte doit être jeté. <img src="/files/ftp_upload/2559/2559fig4.jpg" alt="Figure 4" /> Figure 4. cultures cellulaires mStrawberry OP9. (A) mStrawberry OP9 cellules sont repiquées à70-90% de confluence. (B) Pour une différenciation appropriée, les cellules ES et ES dérivées descendance sont placés sur une couche excessivement confluent de mStrawberry OP9 cellules. <img src="/files/ftp_upload/2559/2559fig5.jpg" alt="Figure 5" /> Figure 5. Représentant ES / mStrawberry profil OP9 flux de co-culture. Toute la descendance ES dérivées de cellules négatives sont mStrawberry et peuvent être triées à partir du flux de OP9 mStrawberry cellules. mStrawberry porte de cellule négative a été déterminée en utilisant un ES/OP9 traditionnelle de co-culture.

<ol>
	<li>Baron, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origins+of+mammalian+hematopoiesis.">Embryonic origins of mammalian hematopoiesis.</a> Exp Hematol. 31, 1160-1160 (2003).</li><li>Baron, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+patterning+of+the+mouse+embryo:+implications+for+hematopoietic+commitment+and+differentiation.">Early patterning of the mouse embryo: implications for hematopoietic commitment and differentiation.</a> Exp Hematol. 33, 1015-1015 (2005).</li><li>Baron, M. H., Fraser, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specification+of+early+hematopoiesis+in+the+mammal.">The specification of early hematopoiesis in the mammal.</a> Curr Opin Hematol. 12, 317-317 (2005).</li><li>Desbaillets, I., Ziegler, U., Groscurth, P., Gassmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryoid+bodies:+an+in+vitro+model+of+mouse+embryogenesis.">Embryoid bodies: an in vitro model of mouse embryogenesis.</a> Exp Physiol. 85, 645-645 (2000).</li><li>Ganapati, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+notch+signaling+in+normal+and+neoplastic+hematopoiesis:+global+gene+expression+profiling+in+response+to+activated+notch+expression.">Modeling notch signaling in normal and neoplastic hematopoiesis: global gene expression profiling in response to activated notch expression.</a> Stem Cells. 25, 1872-1872 (2007).</li><li>Hogan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo : a laboratory manual. , (1994).</li><li>Keller, G., Kennedy, M., Papayannopoulou, T., Wiles, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+commitment+during+embryonic+stem+cell+differentiation+in+culture.">Hematopoietic commitment during embryonic stem cell differentiation in culture.</a> Mol Cell Biol. 13, 473-473 (1993).</li><li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Curr Opin Cell Biol. 7, (1995).</li><li>Nakano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphohematopoietic+development+from+embryonic+stem+cells+in+vitro.">Lymphohematopoietic development from embryonic stem cells in vitro.</a> Semin Immunol. 7, 862-862 (1995).</li><li>Nakano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+development+of+hematopoietic+system+from+mouse+embryonic+stem+cells:+a+new+approach+for+embryonic+hematopoiesis.">In vitro development of hematopoietic system from mouse embryonic stem cells: a new approach for embryonic hematopoiesis.</a> Int J Hematol. 65, 1-1 (1996).</li><li>Nakano, T., Kodama, H., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+lymphohematopoietic+cells+from+embryonic+stem+cells+in+culture.">Generation of lymphohematopoietic cells from embryonic stem cells in culture.</a> Science. 265, 1098-1098 (1994).</li><li>Turksen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Embryonic stem cells : methods and protocols. , (2002).</li></ol>

Une différenciation adéquate des cellules ES sur couche confluente de cellules OP9 mStrawberry résultats dans la production de FLK1 hémangioblastes + au jour 5 de la co-culture. Hémangioblastes doit apparaître comme entassés verticilles de cellules, comme observé dans la figure 1. Pour le reste de la co-culture, visible ES dérivées de la descendance doit apparaître comme des amas de cellules hématopoïétiques (figure 1). Au Jour6 / 7 de deux à quatre groupes de cellules doit apparaître et se développer en des amas de cellules plus grandes (deux adhérentes et en suspension) par jour 8 / 9 de la co-culture. Descendances ES Autres dérivés sont également trouvés étroitement liés dans la couche de cellules stromales adhérentes. Bien que ne figurant pas dans le protocole, il est essentiel de pré-test du FBS utilisée dans le milieu de croissance ES, le OP9 mStrawberry milieux de croissance et de l&#39;OP9 ES / mStrawberry médias différenciation. Correctement testé le sérum doit assurer une bonne croissance des cellules mStrawberry OP9, ainsi que la différenciation correcte des cellules ES en co-culture jusqu&#39;au 5ème jour de la différenciation. Comme alternative ou en complément de mStrawberry OP9 cellules, les cellules OP9 peut être irradié, à 8000 rads, avant de semer à confluence (7.8x10 4 cellules / cm 2). L&#39;irradiation des cellules OP9 permet l&#39;élimination des étapes répétées replating, ainsi que, permet d&#39;éliminer presque complète des anciens, contaminant OP9 cellules à partir d&#39;essais en aval. OP9 cellules ont été conçues pour exprimer la protéine de transduction par mStrawberry OP9 cellules avec 3ug/mL d&#39;un vecteur lentiviral mStrawberry exprimer sous le contrôle d&#39;un promoteur CMV (CMV pRRLsin-vecteur, vecteur de l&#39;UCLA de base). Standard ES/OP9 co-culture protocoles impliquent la mise en place des cellules ES sur une couche confluente OP9 cellules, ainsi que, une étape replating jour 5, afin de réduire ancienne, contaminant OP9 cellules de passage cellulaire. En outre, seuls les cellules hématopoïétiques trouve en suspension sont éliminées pour l&#39;évaluation des ES dérivées de la descendance. Cependant, à la fois avec une étape replating et la récolte des cellules en suspension que l&#39;on manque potentiellement un nombre substantiel de l&#39;élaboration ES dérivées de cellules hématopoïétiques. Cette question devient importante pour répondre lorsque vous essayez de caractériser les défauts hématopoïétiques associés aux lignes ES KO. Afin de contourner ce problème notre laboratoire a utilisé une modification de co-culture, à travers l&#39;utilisation de mStrawberry exprimant OP9 cellules. Ceci assure que toute la descendance ES sont transférés à la poursuite de co-culture au jour 5, et pour la récolte de toute la descendance ES dérivées pour les dosages en aval. Cette modification prévoit une outils utiles dans l&#39;évaluation et la caractérisation des descendances hématopoïétiques provenant à la fois humaines et des lignées ES de souris. Cette OP9 ES-mStrawberry récapitule modèle de co-culture des différentes étapes primitives et l&#39;hématopoïèse définitive. En étudiant les premières étapes du développement hématopoïétique, de nombreuses personnes utilisent le BL-CFC (Blast-comme des cellules formant colonie) de dosage, qui évaluent le développement de l&#39;hémangioblaste des cellules ES. Alors que le test BL-CFC est un outil utile lors des enquêtes début de développement hématopoïétique, le mStrawberry-ES systèmes de co-culture des expositions d&#39;utilité augmenter car elle permet de l&#39;évaluation de l&#39;hémangioblaste, ainsi que, plus adulte lignées hématopoïétiques. Des travaux antérieurs utilisant OP9/ES traditionnelle et des protocoles de différenciation EB a montré que des facteurs supplémentaires peut être nécessaire, comme la surexpression de HOXB4, afin d&#39;en tirer à long terme repeupler les cellules souches hématopoïétiques in vitro. Des travaux supplémentaires sont nécessaires pour évaluer si mStrawberry négatif, triés ES dérivées de populations contiennent vraie cellules souches hématopoïétiques.

OP-9 Cellule Composants Maintenance Medium Stock <table border="1"><tbody><tr><td> Nom de réactif </td><td> Société </td><td> Nombre de catalogue </td><td> Commentaires </td></tr><tr><td> AMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Omega scientifique </td><td> FB01 </td><td> Pré-testés </td></tr><tr><td> L-glutamine </td><td> Cellgro </td><td> 25 à 005-CI </td><td> 200nm </td></tr><tr><td> Trypsine-EDTA </td><td> Stem Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table> ES/OP-9 cellulaire Composants Différenciation moyen Stock <table border="1"><tbody><tr><td> Nom de réactif </td><td> Société </td><td> Nombre de catalogue </td><td> Commentaires </td></tr><tr><td> AMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070 </td><td> Pré-testés </td></tr><tr><td> L-glutamine </td><td> Cellgro </td><td> 25 à 005-CI </td><td> 200 mM </td></tr><tr><td> PBS (sans Ca 2 + et Mg 2 +) </td><td> Cellgro </td><td> 21 à 031-CV </td><td></td></tr><tr><td> Trypsine-EDTA </td><td> Stem Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table>

modified es / op9 co-culture protocol provides enhanced characterization of hematopoietic progeny

La différenciation in vitro des cellules ES vers un destin de cellules hématopoïétiques est utile lorsque l&#39;on étudie les populations de cellules qui sont difficiles d&#39;accès in vivo et pour caractériser les premiers gènes impliqués dans l&#39;hématopoïèse, sans avoir à traiter avec lethalities embryonnaire. La co-culture ES/OP9 système a été initialement conçu pour produire une descendance hématopoïétiques, sans la production au cours des macrophages, comme la ligne OP9 cellules stromales est dérivé de la calvaria de souris mutantes qui manquent ostéopétrose fonctionnelle M-CSF. In vitro, la co-culture ES/OP9 système peut être utilisé afin de récapituler le développement précoce hématopoïétiques. Lorsqu&#39;elle est cultivée sur cellules stromales OP9, les cellules ES se différencient en Flk-1 + hémangioblastes, les progéniteurs hématopoïétiques, et enfin mature, en phase terminale lignées différenciées. La norme ES/OP9 protocole de co-culture implique le placement de cellules ES sur une couche confluente de cellules OP9, ainsi que, périodiquement étapes replating afin d&#39;enlever les vieux, contaminant OP9 cellules. Par ailleurs, les protocoles actuels consistent à évaluer uniquement les cellules hématopoïétiques trouve en suspension et ne sont pas optimisés pour l&#39;évaluation des ES dérivées progéniture à chaque jour de la différenciation. Cependant, avec des étapes replating et la récolte des cellules en suspension que l&#39;on manque potentiellement une grande partie des ES dérivées de la descendance et le développement de cellules hématopoïétiques. Cette question devient importante pour répondre lorsque vous essayez de caractériser les défauts hématopoïétiques associés aux lignes ES KO. Nous décrivons ici une modification ES / mStrawberry OP9 co-culture, qui permet l&#39;élimination des cellules contaminantes OP9 à partir d&#39;essais en aval. Cette méthode permet à l&#39;évaluation complète de tous les descendants ES dérivées à tous les jours de co-culture, résultant en un modèle de différenciation hématopoïétique, qui concernent plus directement le modèle correspond à la différenciation hématopoïétique observées au sein de l&#39;embryon.

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Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

mStrawberry OP9 cellules de permettre une évaluation complète de tous les descendants issus de ES-co-culture.

Modification ES / OP9 co-culture protocole prévoit caractérisation améliorée de Progeny hématopoïétiques

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

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20.6K vues.  University of California, Los Angeles. mStrawberry OP9 cellules de permettre une évaluation complète de tous les descendants issus de ES-co-culture.

Vidéo: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

La congélation des cellules ES humaines

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

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Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Les cellules ES humaines: la culture à partir de cellules congelées

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES Cell dérivé des cultures cellulaires neuroépithéliales

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagation des souches embryonnaires humaines (ES) Cellules

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un compartiment Multi-SNC neurone-glie co-culture de la plate-forme microfluidique

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidiques Co-culture de cellules épithéliales et des bactéries d&#39;enquête sur les Soluble signal médiée par des interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolement des cellules épithéliales mammaires à partir de trois dimensions de cellules mixtes Sphéroïde Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Sous-clonage plus insertion (SPI) - Un Recombineering nouveau procédé pour la construction rapide de ciblage de gène Vecteurs

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Développement d&#39;un système d&#39;insertion Co-culture de deux types cellulaires en l&#39;absence de Cell-Cell Contactez

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...