Ringraziamo U. Ganapati per la creazione e il collaudo dei mStrawberry OP9 linea cellulare. Inoltre, ringraziamo H. Shafifor suo aiuto con la co-coltura. HP è supportato da una borsa di studio presso l&#39;Istituto National Heart, Lung, and Blood (F31HL087714) (HP). Il contenuto di questo lavoro è di esclusiva responsabilità dell&#39;autore e non rappresenta necessariamente il punto di vista ufficiale dell&#39;Istituto National Heart, Lung, and Blood o il National Institutes of Health (NIH). Il meccanismo di Citometria a flusso UCLA Core è supportato dal NIH (CA-16042-28697 e AI), la Jonsson Cancer Center, la UCLA AIDS Institute, e la UCLA School of Medicine.

1. Preparazione di cellule ES <ol><li> Coltura delle cellule ES in base al protocollo standard per la linea cellulare particolare. Assicurati di preparare le cellule ES abbastanza sul piatto della mStrawberry OP9 cellule per il giorno 0 (cellule ES sarà placcato a 1x10 ^ 3 celle per cm 2 al giorno 0) </li></ol> 2. Preparazione delle cellule OP9 <ol><li> Prima di crescita mStrawberry OP9 cellule, preparare il terreno di coltura OP9 OP-9 celle Media Cultura <table border="1"><tbody><tr><td></td><td> Stock </td><td> Conc finale. </td><td> Volume (ml) </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS (OP-9 testato) </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-glutammina </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> Conservare a 4 ° C e utilizzare entro una settimana di preparazione </li><li> Al fine di mantenere mStrawberry OP9 cellule, i media devono essere cambiati ogni 2 giorni e le cellule devono essere subcoltura prima di raggiungere le cellule confluenza (sottocultura al 70-90% confluenza). mStrawberry OP9 colture cellulari non sono mai cresciuta oltre il 90% confluenza come questo si tradurrà in loro differenziazione in adipociti. Vedere la Figura 3. <ol class="lalpha"><li> Cellule subcultura da parte delle cellule 2X lavaggio con PBS (o 1x con PBS + 1x 0,1% con tripsina-EDTA) </li><li> Aggiungi preriscaldata 0,1% tripsina-EDTA a 37 ° C per 5 minuti (o fino a staccare le cellule) </li><li> Quando le cellule sono staccati, aggiungere OP-9 mezzo di crescita. Trasferire le cellule in una provetta da 50mL conica. </li><li> Cellule pellet a 300 xg per 5 minuti a temperatura ambiente. Gettare il surnatante. </li><li> Risospendere le cellule in fresco mStrawberry OP9 terreni di crescita </li><li> Cellule piastra ad una densità minima di 10 4 cellule / cm 2 </li><li> Crescere mStrawberry OP9 cellule a confluenza a partire dal 3 giorni prima del giorno necessario per co-coltura di cellule staminali embrionali (giorno 0, 5 ° giorno, o 8 ° giorno / 9 dell&#39;esperimento) </li></ol></li></ol> 3. ES / mStrawberry OP9 Co-cultura <ol><li> Preparare Co-cultura mediatica differenziazione <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-glutammina </td><td> 200mm </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 cellule (Giorno -3) <ol class="lalpha"><li> SEMPRE Subculture OP9 celle -3 ° giorno, in modo da avere strati confluenti di cellule mStrawberry OP9 per 0 Giornata di co-coltura. Vedi Figura 4b. Piastra mStrawberry OP9 cellule a 1x10 4 / cm 2 per confluenza il giorno 0. Assicurati di sottocultura cellule extra per giorno Giorno 5 e 8 / 9 per la prosecuzione del co-coltura. </li></ol></li><li class="jove_content"> ES / mStrawberry OP9 co-coltura Set-Up (giorno 0) <ol class="lalpha"><li class="jove_content"> Lavare le cellule ES 2 volte con PBS </li><li> Trypsinize cellule ES con pre-riscaldato 0,25% tripsina-EDTA, incubare per 5 min @ 37 ° C </li><li> Aggiungi Co-cultura mediatica differenziazione di cellule </li><li> Pellet di cellule staminali a 400xg, 5-7min, RT. Gettare il surnatante </li><li> Risospendere le cellule in co-cultura mediatica differenziazione </li><li> Rimuovere i supporti da mStrawberry OP9 cellule </li><li> Aggiungi Co-cultura mediatica differenziazione mStrawberry fiaschi OP9 </li><li> Piastra 1x10 3 celle ES / cm 2 a fiasco di mStrawberry OP9 cellule. Incubare le cellule a 37 ° C, 5% di CO 2 con l&#39;umidità. </li></ol></li><li> mStrawberry OP9 cellule (Giorno 2) <ol class="lalpha"><li> MStrawberry sottocultura OP9 in modo da avere strati confluenti di cellule mStrawberry OP9 per il giorno 5 di co-coltura. Piastra mStrawberry OP9 cellule a 1x10 4 / cm 2 per confluenza in occasione della Giornata 5 (come al punto 3.2). Assicurati di sottocultura celle supplementari per il giorno 8 / 9, se necessario (come al punto 2.2). </li></ol></li><li> ES / mStrawberry OP9 co-coltura (Giorno 3) <ol class="lalpha"><li> Rimuovere con attenzione medio di co-coltura e sostituire con nuovi co-cultura medio differenziazione </li></ol></li><li> ES / mStrawberry OP9 co-coltura (giorno 5) <ol class="lalpha"><li> Lavare accuratamente co-colture 2x con PBS </li><li> Trypsinize cellule con pre-riscaldato 0,25% tripsina-EDTA </li><li> Lavare le cellule off pallone con co-cultura mediatica differenziazione </li><li> Celle a pellet 400xg, 7min, RT. Gettare il surnatante </li><li>Aggiungi Co-cultura mediatica differenziazione di cellule. Utilizzare una 21-gauge bluntended per risospendere le cellule e per garantire la sospensione singola cella </li><li> Contare le cellule utilizzando un emocitometro. Dovrebbe avere 100-150 volte maggiore del numero di cellule ES da giorno 0. </li><li> Re-seme 6.48x10 4 / cm 2 - 7.76x10 4 / cm 2 co-coltura le cellule su un nuovo livello di confluenti, mStrawberry OP9 cellule </li><li> Giorno 5 TEST-rimanente co-coltura le cellule possono essere utilizzate per l&#39;analisi di citometria a flusso, cytopreps (30K celle), le analisi ChIP, ecc </li></ol></li><li> ES / mStrawberry OP9 co-coltura (giorno 8 / 9 e Giorno 12) <ol class="lalpha"><li> Rimuovere i supporti di differenziazione dal pallone e salvare. Lavare monostrato 1x con PBS per rimuovere le cellule ematopoietiche in sospensione. Non gettare le cellule ematopoietiche. </li><li> Trypsinize cellule aderenti con pre-riscaldato 0,25% tripsina-EDTA </li><li> Lavare le cellule off pallone con i media differenziazione ES e aggiungere alle cellule ematopoietiche si trovano in sospensione = </li><li> Celle a pellet 400xg, 7min, RT. Gettare il surnatante </li><li> Aggiungi Co-cultura differenziazione dei media o PBS alle cellule. Utilizzare una 21-gauge bluntended per risospendere le cellule e di assicurare per le singole cellule delle cellule sospensione Conte utilizzando un emocitometro. </li><li> Co-coltura può essere continuato per 14 giorni per valutare progenitori emopoietici più maturo da risemina co-coltura le cellule su un nuovo livello di confluenti, mStrawberry OP9 cellule in occasione della Giornata 8 o 9 e poi di nuovo il giorno 12. Re-seed numero di cellulare ottimizzato basato sul recupero delle cellule necessarie per giorno di test. </li></ol></li><li> Valutazione della ES-derivati ​​progenie (giorno 1-14). ES-derivati ​​progenie possono essere valutate in qualsiasi giorno della co-coltura di flusso smistamento mStrawberry cellule negative (ES-derivati ​​progenie). <ol class="lalpha"><li> Rimuovere i supporti di differenziazione dal pallone e salvare. Lavare monostrato 1x con PBS per rimuovere le cellule ematopoietiche in sospensione. Non gettare le cellule ematopoietiche. </li><li> Trypsinize cellule aderenti con pre-riscaldato 0,25% tripsina-EDTA </li><li> Lavare le cellule off pallone con co-cultura mediatica differenziazione e aggiungere alle cellule ematopoietiche si trovano in sospensione </li><li> Celle a pellet 400xg, 7min, RT. Gettare il surnatante </li><li> Risospendere le cellule con 21-gauge bluntended e contare </li><li> Progenie ES-derivati ​​(mStrawberry cellule negative) possono essere ordinati dal flusso positivo mStrawberry OP9 cellule. Progenie ES-derivati ​​possono essere caratterizzati con l&#39;analisi in citometria a flusso, cytopreps, analisi ChIP, ecc </li></ol></li></ol> 4. Rappresentante dei risultati: <img src="/files/ftp_upload/2559/2559fig1.jpg" alt="Figura 1" /> Figura 1. ES / mStrawberry OP9 co-cultura di sistema. Il nostro laboratorio utilizza un cellulare in vitro in ES co-coltura di sistema per lo sviluppo del modello ematopoietiche. In questo sistema di co-coltura, le cellule ES differentiate, in emangioblasti al 5 ° giorno, quando si trova su uno strato confluente di mStrawberry OP9 cellule. Come co-coltura procede, precursori ematopoietici sono presenti 8 ° giorno, mentre terminalmente differenziate linee ematopoietiche appaiono di giorno 14. Le frecce indicano i giorni di differenziazione quando passaging di ES-derivati ​​progenie sui nuovi strati confluenti di mStrawberry OP9 cellule necessarie. <img src="/files/ftp_upload/2559/2559fig2.jpg" alt="Figura 2" /> Figura 2. Una corretta differenziazione delle cellule ES. Le cellule staminali sono seminate su uno strato confluente di mStrawberry OP9 il giorno 0. ES-derivati ​​progenie cominciano a diventare visibili a 3 ° giorno di differenziazione, con ES-derivati ​​progenie che sembra un ammasso di cellule con un bordo definito o iniziando a formare un modello spirale. Emangioblasti, che sono i precursori comuni mesoderma per i lignaggi endoteliali e ematopoietiche, dovrebbe apparire come ammucchiate spirali di cellule al 5 ° giorno. Per la co-coltura periodi superiori a cinque giorni, ES-derivati ​​progenie dovrebbe apparire come ammassi di cellule ematopoietiche. Da due a quattro gruppi monocellulari, in occasione della Giornata 6 / 7, dovrebbe comparire e crescere in gruppi di cellule più grandi entro il giorno 8 / 9 della co-coltura. Progenie ES altri derivati ​​si trovano anche strettamente legata allo strato aderente cellule stromali. <img src="/files/ftp_upload/2559/2559fig3.jpg" alt="Figura 3" /> Figura 3. Infruttuoso differenziazione delle cellule ES. I risultati di questa co-coltura riflettere improprio differenziazione delle cellule ES. Si noti come l&#39;ES-derivati ​​progenie non appaiono in un modello a spirale 5 ° giorno. L&#39;assenza di cellule in un modello spirale indica la formazione impropria hemangioblast. Se hemangioblast non si sviluppano correttamente, poi continuò co-coltura si tradurrà in stentato differenziazione lignaggio ematopoietiche. Co-culture con formazione hemangioblast improprio devono essere eliminati. <img src="/files/ftp_upload/2559/2559fig4.jpg" alt="Figura 4" /> Figura 4. mStrawberry OP9 colture cellulari. (A) mStrawberry cellula OP9 sono subcoltura a70-90% confluenza. (B) Per la differenziazione corretta, le cellule ES e ES-derivati ​​progenie sono posti su uno strato troppo confluenti di mStrawberry OP9 cellule. <img src="/files/ftp_upload/2559/2559fig5.jpg" alt="Figura 5" /> Figura 5. Rappresentante ES / mStrawberry OP9 Co-cultura profilo di flusso. Tutti ES-derivati ​​progenie sono mStrawberry cellule negative e possono essere ordinati dal flusso mStrawberry OP9 cellule. mStrawberry porta cellulare negativa è stata determinata utilizzando un tradizionale ES/OP9 co-coltura.

<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>

Nessun conflitto di interessi dichiarati.

Corretta differenziazione delle cellule ES sullo strato confluenti di cellule mStrawberry OP9 risultati nella produzione di Flk1 emangioblasti + al 5 ° giorno di co-coltura. Emangioblasti dovrebbe apparire come ammucchiate spirali di cellule, come osservato in Figura 1. Per il resto della co-coltura, visibile ES-derivati ​​progenie dovrebbe apparire come ammassi di cellule ematopoietiche (Figura 1). Al 6 ° giorno / 7 03:58 ammassi cellulari dovrebbe comparire e crescere in cluster cella più grande (sia aderenti e in sospensione) entro il giorno 8 / 9 della co-coltura. Progenie ES altri derivati ​​si trovano anche strettamente legati all&#39;interno dello strato aderente cellule stromali. Anche se non elencati nel protocollo, è fondamentale pre-test del FBS utilizzare nel supporto della crescita ES, la mStrawberry OP9 terreni di coltura e la ES / mStrawberry OP9 mezzi differenziazione. Adeguatamente testati siero dovrebbe garantire la corretta crescita di mStrawberry OP9 cellule, così come, proprio la differenziazione delle cellule ES in co-coltura fino a 5 ° giorno di differenziazione. In alternativa o in aggiunta al mStrawberry OP9 cellule, OP9 cellule possono essere irradiati, a 8000 rad, prima della semina a confluenza (7.8x10 4 celle / cm 2). Irradiazione di cellule OP9 permette l&#39;eliminazione delle ripetute fasi replating, nonché, permette l&#39;eliminazione quasi totale della vecchia, contaminando OP9 cellule di test a valle. OP9 cellule sono state progettate per esprimere la proteina mStrawberry di trasduzione OP9 cellule con 3ug/mL di un mStrawberry vettori lentivirali che esprimono sotto il controllo di un promotore CMV (pRRLsin-CMV vettore, UCLA vettore core). Standard ES/OP9 co-coltura protocolli comportano il collocamento di cellule staminali embrionali su un confluenti strato OP9 cellule, così come, a 5 passo replating giorno per ridurre vecchio, contaminando OP9 cellule dal passaggio delle cellule. Inoltre, solo le cellule ematopoietiche si trovano in sospensione vengono rimossi per la valutazione di ES-derivati ​​progenie. Tuttavia, sia con un passo replating e la raccolta di cellule in sospensione una sola manca potenzialmente un numero considerevole di sviluppo ES-derivato cellule ematopoietiche. Questo problema diventa importante affrontare quando si cerca di caratterizzare i difetti ematopoietiche associati a linee ES eliminazione diretta. Per aggirare questo problema il nostro laboratorio utilizzato una versione modificata di co-cultura, attraverso l&#39;utilizzo di mStrawberry che esprimono OP9 cellule. Questo assicura che tutte le progenie ES sono trasferite al continuo co-cultura al giorno 5, e per la raccolta di tutte le ES-derivati ​​progenie per saggi a valle. Questa modifica fornisce strumenti utili nella valutazione e caratterizzazione di progenie ematopoietiche derivate da entrambe le ES umane e delle linee del mouse. Questo ES-mStrawberry OP9 co-coltura modello ricapitola le varie fasi primitive e ematopoiesi definitiva. Nello studiare le prime fasi di sviluppo ematopoietici, molte persone utilizzano la batteria BL-CFC (Blast-come cellule formanti colonie) test, che valutano lo sviluppo del hemangioblast dalle cellule staminali. Mentre il modello BL-CFC saggio è uno strumento utile nell&#39;ambito delle indagini primi stadi dello sviluppo ematopoietico, il mStrawberry-ES co-coltura sistemi mostre utilità aumentare in quanto consente per la valutazione delle hemangioblast, così come, linee ematopoietiche più adulto. Il lavoro precedente con OP9/ES tradizionali e protocolli differenziazione EB ha dimostrato che altri fattori forse necessario, come l&#39;iperespressione di HoxB4, al fine di ricavare lungo termine ripopolare le cellule staminali ematopoietiche in vitro. Lavoro supplementare è necessario valutare se mStrawberry negativo, in ordine ES-derivati ​​popolazioni contengono vere cellule staminali ematopoietiche.

OP-9 celle di manutenzione Media Archivi Componenti <table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> aMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Omega scientifico </td><td> FB01 </td><td> Pre-Tested </td></tr><tr><td> L-glutammina </td><td> Cellgro </td><td> 25-005-CI </td><td> 200nm </td></tr><tr><td> Tripsina-EDTA </td><td> Stem Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table> ES/OP-9 differenziazione delle cellule Media Archivi Componenti <table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </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> Pre-Tested </td></tr><tr><td> L-glutammina </td><td> Cellgro </td><td> 25-005-CI </td><td> 200mm </td></tr><tr><td> PBS (senza Ca 2 + e Mg 2 +) </td><td> Cellgro </td><td> 21-031-CV </td><td></td></tr><tr><td> Tripsina-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 differenziazione in vitro di cellule staminali verso un destino di cellule ematopoietiche è utile quando si studiano popolazioni di cellule che sono di difficile accesso in vivo e per caratterizzare i primi geni coinvolti nella ematopoiesi, senza avere a che fare con lethalities embrionale. Il ES/OP9 co-coltura sistema è stato originariamente progettato per produrre progenie ematopoietiche, senza la produzione su dei macrofagi, come il OP9 linea di cellule stromali deriva dal calvaria di topi mutanti privi di osteopetrosi funzionale M-CSF. In vitro ES/OP9 co-coltura sistema può essere utilizzato al fine di ricapitolare primo sviluppo ematopoietiche. Quando OP9 coltivati ​​su cellule stromali, cellule staminali si differenziano in Flk-1 + emangioblasti, progenitori emopoietici, e finalmente maturo, lignaggi terminalmente differenziate. Lo standard ES/OP9 co-coltura protocollo prevede l&#39;inserimento di cellule staminali embrionali su uno strato confluente di OP9 cellule, così come, a pochi passi replating periodici al fine di rimuovere vecchi, contaminando OP9 cellule. Inoltre, i protocolli attuali coinvolgono valutare solo le cellule ematopoietiche si trovano in sospensione e non sono ottimizzati per la valutazione di ES-derivati ​​della progenie per ogni giorno di differenziazione. Tuttavia, con passi replating e la raccolta di cellule in sospensione una sola manca potenzialmente una grande porzione di ES-derivati ​​progenie e lo sviluppo di cellule ematopoietiche. Questo problema diventa importante affrontare quando si cerca di caratterizzare i difetti ematopoietiche associati a linee ES eliminazione diretta. Qui si descrive una versione modificata ES / mStrawberry OP9 co-coltura, che consente l&#39;eliminazione dei contaminanti OP9 cellule di test a valle. Questo metodo permette la valutazione completa di tutti gli ES-derivati ​​della progenie per tutti i giorni di co-cultura, risultando in un modello di differenziazione ematopoietiche, che più direttamente corrisponde al modello di differenziazione ematopoietiche osservate entro l&#39;embrione.

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

mStrawberry OP9 cellule permettere una valutazione completa di tutte le ES-derivati ​​dalla progenie di co-coltura.

Modificato ES / OP9 Co-Cultura Protocollo prevede Caratterizzazione avanzata di Progeny emopoietiche

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 Views.  University of California, Los Angeles. mStrawberry OP9 cellule permettere una valutazione completa di tutte le ES-derivati ​​dalla progenie di co-coltura.

Video: 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.

Congelamento cellule staminali embrionali umane

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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Biologia

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.

Le cellule umane ES: Cultura partire da cellule congelate

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-derivati ​​colture cellulari neuroepiteliale

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.

Propagazione di staminali embrionali umane (ES) Cellule

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 e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

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 multi-vano SNC Neuron-glia Co-cultura Microfluidic piattaforma

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.

Microfluidica Co-coltura di cellule epiteliali e batteri per le indagini solubile del segnale mediato da Interazioni

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.

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid Co-cultura

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

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.

Sviluppo di un sistema di inserimento co-coltura di due tipi cellulari in assenza del cellula-cellula di contatto

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 ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

Protocollo guidata per caratterizzazione microbica fecale mediante sequenziamento del 16S rRNA-amplicone

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...