Un ringraziamento particolare al Dr. Boris Greber per impostare il Activin Un protocollo ELISA pubblicato in Greber et al. 2007. Siamo particolarmente grati alla signora Monica Shevack per preparare la panoramica grafica. Siamo molto grati al dottor Fuchs Heiko per tutto l&#39;aiuto e preziosi consigli prima e durante le riprese. Vorremmo ringraziare tutti i membri del laboratorio Adjaye, soprattutto Elisabeth Socha per mantenere un costante rifornimento di MEF e CM. Riconosciamo anche i nostri colleghi presso l&#39;impianto di animale della MPIMG per il loro sostegno permanente. Questo lavoro è stato parzialmente finanziato dalla Max Planck Society e il [BMBF; numero di concessione 0315717A], partner del ERASysBio + iniziativa finanziata nell&#39;ambito del EU ERA-NET Plus nel 7 ° PQ.

1. Isolamento dei fibroblasti embrionali del mouse (MEF) Le seguenti due fasi vengono eseguite sotto condizioni non asettiche. <ol><li> Sacrifica un topo in stato di gravidanza (CF1, Harlan, USA) a 13 o 14 DPC (giorni post-coitum) per dislocazione cervicale. </li><li> Dissect le corna uterini, sciacquare brevemente nel 70% (v / v) etanolo e posto in una provetta Falcon contenente PBS senza Ca 2 + Mg 2 + (Gibco, Invitrogen). </li></ol> Le seguenti operazioni sono eseguite in una cappa coltura tissutale in condizioni asettiche e utilizzando strumenti sterili. <ol start="3"><li> Mettere le corna uterine in una capsula Petri e separare ogni embrione dalla sua placenta e il sacco embrionale. </li><li> Staccare testa e gli organi rosse, lavare in PBS e mettere tutti gli embrioni in un piatto pulito Petri. Tritare finemente il tessuto usando una lama di rasoio sterile fino diventa possibile pipettare. </li><li> Aggiungere 1 ml di 0,05% tripsina / EDTA (Gibco, Invitrogen), including 100 unità Kunitz di DNasi I (USB), per embrione. </li><li> Trasferire il tessuto in una provetta Falcon da 50 ml e incubare per 15 min a 37 ° C. Dopo ogni 5 minuti di incubazione, le cellule dissociare pipettando su e giù accuratamente. </li><li> Inattivare la tripsina aggiungendo circa 1 volume di terreno MEF appena preparato. MEF terreno di coltura (componenti per fare 500 ml di media, mescolare tutti i componenti e filtro): 450 ml di DMEM, 50 ml di FBS (10% (v / v)), 5 ml di 200 mM L-glutammina (1/100 (v / v)), 5 ml di penicillina-streptomicina (1/100 (v / v)). </li><li> Centrifugare le cellule con bassa velocità (300 xg), 5 min, rimuovere con cautela il surnatante e risospendere il pellet cellulare in media MEF caldo. </li><li> Tavola circa un numero di celle che è equivalente a 3-4 embrioni in ciascun (TPP) pallone T150 rivestiti con 0,2% di gelatina (gelatina di pelle bovina, tipo B, Sigma) per 2 h. I fibroblasti (P0, il passaggio 0) sono le uniche cellule che hanno la possibilità di allegare alla gelatina rivestite fiaschi. </li&gt; <li> Idealmente, le cellule sono confluenti 80-90% dopo 24 ore e in questa fase una parte importante del P0 cellule viene congelato per uso futuro. </li><li> Espandere il restante T150 pallone (s) di P0 cellule fino P3 o P4, e quindi inattivare usare come alimentatori per replate hESC o per produrre mezzo condizionato (CM). </li></ol> 2. Inattivazione e placcatura MEF (Feeder Preparazione Cells) Tutte le fasi sono eseguite in una cappa coltura tissutale in condizioni asettiche. <ol><li> Cappotto T150 palloni con il 0,2% di gelatina ed incubare a temperatura ambiente per almeno 2 ore. </li><li> Diluire mitomicina C in PBS (1 mg / ml) e filtrare. </li><li> Aspirare media da MEF e lavare con PBS senza Ca 2 + Mg 2 +. </li><li> Trasferire 20 ml di mezzo contenente 10 ug / ml di mitomicina C il MEF. </li><li> Incubare per 2 ore a 37 ° C con mitomicina C contenente il mezzo poi lavate due volte con PBS, Tripsinizzare, centrifuga (per 5 minuti a 300 xg) e risospendere le cellule in mezzo caldo. </li><li>Contare le cellule e la piastra ad una densità di 56.000 cellule / cm 2 in fiasche T150 e di utilizzare per la produzione CM per i successivi 6 giorni. </li></ol> 3. Condizionata Medium (CM) Preparazione Tutte le fasi sono eseguite in una cappa coltura tissutale in condizioni asettiche. <ol><li> Il giorno dopo la piastratura inattivato MEF ad una densità di 56.000 cellule / cm 2 sostituire il mezzo MEF con il mezzo hESC (UM, media incondizionata) (0,5 ml / cm 2) appena completato con 4 ng / ml di FGF2. </li><li> Raccogliere CM da beute alimentazione dopo 24 h di incubazione e aggiungere mezzo fresco contenente hESC 4 ng / ml di FGF2 agli alimentatori. </li><li> Ripetere questa procedura per i prossimi 6 giorni. Ogni negozio raccolti CM giorni a -20 ° C. </li><li> Dopo 6 giorni mescolare tutte le aliquote di medio e filtro (Corning, 0,22 micron, PAS). Effettuare aliquote di 50 ml e conservare a -80 ° C. </li><li> Supplemento CM con ulteriori 4 ng / ml di FGF2 prima di aggiungere al hESC coltivate Matrigel. &lt;/ Li&gt; </ol> 4. Misura di Activin A in Media condizionata (ELISA) 15 <ol><li> Portare tutti i campioni e reagenti a temperatura ambiente. </li><li> Diluire la cattura anticorpale (Human \ Mouse \ Rat activina A MAB, R &amp; D Systems) in PBS con 1% BSA, aggiungere alla micropiastra (100 pl / pozzetto) ed incubare una notte a temperatura ambiente. </li><li> Dopo 24 ore lavare i pozzetti tre volte con PBST (PBS con 0,05% Tween 20) (300 pl / pozzetto), allora blocco (1% BSA / PBS, 300 pl / pozzetto) per 1 ora a RT. </li><li> In questo tempo preparare una Activin A (R &amp; D Systems) curva standard, tra cui 7 diluizioni (concentrazione inferiore a 30 ng / ml) e il campione in bianco. Il campo lineare di lavoro del Activin A è tra 0,25 e 32 ng / ml. </li><li> Aggiungi duplicati degli standard e dei campioni ai pozzetti (100 pl / pozzetto) e incubare per 2 ore a temperatura ambiente. </li><li> Lavare tre volte (300 ml di PBST) pozzi. </li><li> Aggiungere il secondario (biotinilato) anticorpi (Human / Mouse / Rat Biotinylated Activin A MAb, R &amp; D Systems) (0.25 ug / ml in 1% BSA / PBS) e incubare per 2 ore a temperatura ambiente. </li><li> Lavare tre volte (300 ml di PBST) pozzi, aggiungere streptavidina-HRP (diluita in 1% BSA / PBS, R &amp; D Systems) e incubare per 20 minuti a RT. </li><li> Lavare tre volte (300 ml di PBST) pozzetti, aggiungere 100 microlitri di soluzione di substrato (Quantikine, R &amp; D Systems) e incubare per 30 min a RT al buio. </li><li> Aggiungere 100 ml di soluzione di arresto (Quantikine, R &amp; D Systems) in ciascun pozzetto e mescolare delicatamente. </li><li> Set Lettore di micropiastre (Molecular Devices Spectra Max 250, Global Medical Instrumentation, Inc., Minnesota) a 450 nm, con correzione lunghezza d&#39;onda a 540 o 570 nm, per determinare la densità ottica di ciascun pozzetto. </li></ol> 5. Risultati rappresentativi Lo schema generale della procedura di isolamento è presentato in Figura 1. La morfologia tipica delle hESC hiPSCs e coltivate in condizioni differenti è presentata in Figura 2. La morfologia dei<html> MEF e cellule feeder inattivate usati per preparare CM è presentato nella Figura 3. In generale, le celle devono essere confluenti 24 ore dopo l&#39;isolamento e pronti per essere congelati o espanso. Tuttavia, a volte potrebbe richiedere 2-3 giorni prima di ottenere colture confluenti. Il CM deve essere preparato dalle cellule al passaggio 4 e non dopo. Questo è fondamentale perché le cellule primarie può essere ampliato per 4-5 passaggi prima dell&#39;inizio della senescenza. La citochina, Activin A, è considerato il principale fattore critico secreto dalle cellule di alimentazione per il sostegno di crescita indifferenziata di cellule pluripotenti 14. La misurazione del livello di Activin A in CM (figura 4) è un saggio quantitativo molto conveniente per monitorare la qualità di MEF. <img alt="Figura 1" src="/files/ftp_upload/3854/3854fig1.jpg" /> Figura 1. Una rappresentazione schematica della procedura di isolamento MEF. ONTENUTO &quot;&gt; <img alt="Figura 2" src="/files/ftp_upload/3854/3854fig2.jpg" /> Figura 2. La morfologia tipica delle hESC indifferenziati coltivate in presenza di (A), le cellule di alimentazione (B) mezzo condizionato e media (C) definita. La morfologia tipica di hiPSCs coltivate in presenza di (D, E) cellule di alimentazione. <img alt="Figura 3" src="/files/ftp_upload/3854/3854fig3.jpg" /> Figura 3. La morfologia tipica di fibroblasti embrionali di topo (MEF). (A) Passaggio 0 (P0) due giorni dopo placcatura / isolamento, (B) inattivato strato alimentatore ad una densità di 56.000 cellule / cm 2. <img alt="Figura 4" src="/files/ftp_upload/3854/3854fig4.jpg" /> Figura 4. Enzyme-linked immunosorbent assay (ELISA) a base di misurazioni della concentrazione di Activin A in mezzo condizionato (CM) prepared con fibroblasti embrionali di topo derivate dal ceppo di topi CF1. CM è stato raccolto per 6 giorni e poi sommati. CM &quot;1&quot; e CM &quot;2&quot; si riferiscono a diversi lotti di media e di messaggistica unificata ai media incondizionati. Poiché la funzione del processo di condizionamento viene Activin A secrezione nel mezzo di MEF, Activin Un quasi impercettibile a UM.

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonally+derived+human+embryonic+stem+cell+lines+maintain+pluripotency+and+proliferative+potential+for+prolonged+periods+of+culture.">Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.</a> Dev. Biol. 227, 271-278 (2000).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Ludwig, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cells+in+defined+conditions.">Derivation of human embryonic stem cells in defined conditions.</a> Nat. Biotechnol. 24, 185-187 (2006).</li><li>Yao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+self-renewal+and+directed+differentiation+of+human+embryonic+stem+cells+in+chemically+defined+conditions.">Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 6907-6912 (2006).</li><li>Lu, J., Hou, R., Booth, C. J., Yang, S. H., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+culture+conditions+of+human+embryonic+stem+cells.">Defined culture conditions of human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 5688-5693 (2006).</li><li>Skottman, H., Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+for+human+embryonic+stem+cells.">Culture conditions for human embryonic stem cells.</a> Reproduction. 132, 691-698 (2006).</li><li>Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., Bongso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeders+support+prolonged+undifferentiated+growth+of+human+inner+cell+masses+and+embryonic+stem+cells.">Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.</a> Nat. Biotechnol. 20, 933-936 (2002).</li><li>Richards, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+various+human+feeders+for+prolonged+undifferentiated+growth+of+human+embryonic+stem+cells.">Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.</a> Stem Cells. 21, 546-556 (2003).</li><li>Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+layers+for+human+embryonic+stem+cells.">Human feeder layers for human embryonic stem cells.</a> Biol. Reprod. 68, 2150-2156 (2003).</li><li>Inzunza, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cell+lines+in+serum+replacement+medium+using+postnatal+human+fibroblasts+as+feeder+cells.">Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells.</a> Stem Cells. 23, 544-549 (2005).</li><li>Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+Human+Amniotic+Mesenchymal+Cells+as+Feeder+Layers+to+Sustain+Propagation+of+Human+Embryonic+Stem+Cells+in+the+Undifferentiated+State.">Utilization of Human Amniotic Mesenchymal Cells as Feeder Layers to Sustain Propagation of Human Embryonic Stem Cells in the Undifferentiated State.</a> Cell Reprogram. , (2011).</li><li>Eiselleova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+mouse+and+human+feeder+cells+for+human+embryonic+stem+cells.">Comparative study of mouse and human feeder cells for human embryonic stem cells.</a> Int. J. Dev. Biol. 52, 353-363 (2008).</li><li>Greber, B., Lehrach, H., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+2+modulates+transforming+growth+factor+beta+signaling+in+mouse+embryonic+fibroblasts+and+human+ESCs+(hESCs)+to+support+hESC+self-renewal.">Fibroblast growth factor 2 modulates transforming growth factor beta signaling in mouse embryonic fibroblasts and human ESCs (hESCs) to support hESC self-renewal.</a> Stem Cells. 25, 455-464 (2007).</li><li>Vallier, L., Alexander, M., Pedersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin/Nodal+and+FGF+pathways+cooperate+to+maintain+pluripotency+of+human+embryonic+stem+cells.">Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells.</a> J. Cell Sci. 118, 4495-4509 (2005).</li></ol>

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La procedura di isolamento MEF qui presentata consente la creazione di una condizione di cultura standardizzata per le hESC e hiPSCs. Inoltre il sistema ELISA basato utilizzato per valutare la produzione di citochine da cellule di alimentazione è un utile indicatore della qualità dei mezzi condizionati MEF-derivati. La manutenzione ordinaria del ceppo di topo (CF1) fornendo fibroblasti supporto è necessaria per evitare lotto a lotto variazione di terreni di coltura. Inoltre, si raccomanda di isolare embrioni di topo contemporaneamente per ottenere una qualità costante di cellule. Certo, appena isolato MEF può essere mantenuto congelato a P0 e P1. Inoltre inattivati ​​MEF può essere conservato congelato in aliquote di quantità appropriate a seconda delle esigenze per colture cellulari. Di solito circa 250.000 cellule devono essere seminati in un singolo pozzo di una piastra da 6 pozzetti di hESC cultura e le cellule iPS. Il metodo consolidato e ottimizzato può essere utilizzato di routine per ridurre la variabilità tra diffeaffitto esperimenti.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> DMEM (glucosio) </td><td> Gibco, Invitrogen </td><td> 41966-052 </td><td></td></tr><tr><td> FBS </td><td> Biochrom </td><td> S0115 </td><td></td></tr><tr><td> L-glutammina </td><td> Gibco, Invitrogen </td><td> 25030-024 </td><td></td></tr><tr><td> Penicillina-streptomicina </td><td> Gibco, Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Sistema di aspirazione del filtro </td><td> Corning </td><td> 431097 </td><td> 500 ml, 0,22 micron, PAS </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650 </td><td></td></tr><tr><td> Knockout DMEM </td><td> Gibco, Invitrogen </td><td> 10829-018 </td><td></td></tr><tr><td> Knockout Serum sostituzione </td><td> Gibco, Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> Aminoacidi non essenziali </td><td> Gibco, Invitrogen </td><td> 11140-035 </td><td></td></tr><tr><td> beta-mercaptoetanolo </td><td> Sigma </td><td> M7522 </td><td></td></tr><tr><td> PBS </td><td> Gibco, Invitrogen </td><td> 14190-169 </td><td></td></tr><tr><td> DNasi I </td><td> Sigma </td><td> D4527 </td><td></td></tr><tr><td> Mitomicina C </td><td> Roche </td><td> 10107409001 </td><td></td></tr><tr><td> Base Fibroblast Growth Factor </td><td> Peprotech </td><td> 100-18B </td><td></td></tr><tr><td> Biotinilato Anti-human/mouse/rat Activin Un anticorpo </td><td> R &amp; D Systems </td><td> BAM3381 </td><td></td></tr><tr><td> Gelatina </td><td> Sigma </td><td> G9391 </td><td></td></tr><tr><td> Umano / Mouse /Rat Activin A MAb </td><td> R &amp; D Systems </td><td> MAB3381 </td><td></td></tr><tr><td> 0,05% tripsina-EDTA </td><td> Gibco, Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> Extra Scissors Iris sottili </td><td> FST </td><td> 14088-10 </td><td></td></tr><tr><td> Extra Pinza Graefe Belle </td><td> FST </td><td> 11150-10 </td><td></td></tr><tr><td> Pinza di fissaggio Pierse </td><td> FST </td><td> 18155-13 </td><td></td></tr></tbody></table>

preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells

In generale, le cellule staminali embrionali umane (hESC) e umani cellule staminali pluripotenti indotte (hiPSCs) 1 può essere coltivato in condizioni variabili. Tuttavia, non è facile stabilire un sistema efficace per la coltura di tali cellule. Poiché le condizioni di coltura può influenzare l&#39;espressione del gene che conferisce la pluripotenza in hESC e hiPSCs, l&#39;ottimizzazione e la standardizzazione del metodo di cultura è fondamentale. La creazione di linee di hESC è stato descritto utilizzando MEF come cellule di alimentazione e siero bovino fetale (FBS)-2 contenente terreno di coltura. Successivamente, FBS è stato sostituito con la sostituzione knockout siero (KSR) e FGF2, che aumenta la proliferazione delle hESC 3. Infine, l&#39;alimentatore senza sistemi di coltura permettono alle cellule in coltura Matrigel rivestite con lastre in KSR contenente mezzo condizionato (medium condizionato dal MEF) 4. Successivamente, condizioni di coltura hESC si sono spostati verso alimentatore privo di cultura nel definire chimicamenteEd condizioni 5-7. Inoltre, per evitare il potenziale contaminazione da agenti patogeni e proteine ​​animali metodi di coltura utilizzando xeno-free componenti sono state stabilite 8. Per ottenere migliori condizioni cellule di alimentazione di topo sono stati sostituiti con linee cellulari umane (ad esempio muscolari fetale e 9 cellule della pelle, cellule adulte 10, fibroblasti prepuzio 11-12, amniotico cellule mesenchimali 13). Tuttavia, l&#39;efficienza di mantenere hESC indifferenziate utilizzando prepuzio fibroblasti umani di derivazione strati di alimentazione non è così elevata come quella di cellule feeder topo a causa del basso livello di secrezione di Activin A 14. Ovviamente, vi è una differenza evidente nella produzione di fattore di crescita di topo e cellule feeder umane. L&#39;analisi dei trascrittomi di topo e cellule di alimentazione umana ha rivelato differenze significative tra le cellule di sostegno e non di sostegno. Esogena FGF2 è fondamentale per eseguire manutenzioneNing auto-rinnovamento delle hESC e hiPSCs, ed è stato identificato come un fattore chiave regolando l&#39;espressione di Tgfβ1, Activin A e Gremlin (un antagonista BMP) nelle cellule di alimentazione. Activin A ha mostrato di indurre l&#39;espressione di OCT4, SOX2, e Nanog in hESC 15-16. Per lungo termine cultura, hESC e hiPSCs possono essere coltivate su mitoticamente inattivato MEF o sotto alimentatore senza condizioni MEF-CM (MEF condizionata Medium) su Matrigel rivestite con lastre di mantenere il loro stato indifferenziato. Il successo di entrambe le condizioni di coltura dipende completamente la qualità delle cellule di alimentazione, poiché influenza direttamente la crescita delle hESC. Qui, presentiamo un metodo ottimizzato per l&#39;isolamento e la coltura di fibroblasti embrionali di topo (MEF), preparazione del terreno condizionato (CM) e enzyme-linked test immunoenzimatico (ELISA) per valutare i livelli di Activin A all&#39;interno dei media.

Watch this Scientific Journal Video about Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells at JoVE.com

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

La qualità di fibroblasti embrionali di topo (MEF) è dettata dal ceppo destro del mouse, ad esempio CF-1. Pluripotenza-solidale MEF e mezzi condizionati (CM) ottenuti da questi dovrebbe contenere le concentrazioni ottimali di Activin A, Gremlin e Tgfβ1 necessari per la Activin / Nodale FGF e percorsi di co-operativamente mantenere il self-renewal e la pluripotenza.

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

preparation-mouse-embryonic-fibroblast-cells-suitable-for-culturing

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90.4K Views.  Max Planck Institute for Molecular Genetics. La qualità di fibroblasti embrionali di topo (MEF) è dettata dal ceppo destro del mouse, ad esempio CF-1. Pluripotenza-solidale MEF e mezzi condizionati (CM) ottenuti da questi dovrebbe contenere le concentrazioni ottimali di Activin A, Gremlin e Tgfβ1 necessari per la Activin / Nodale FGF e percorsi di co-operativamente mantenere il self-renewal e la pluripotenza.

Video: Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagazione di staminali embrionali umane (ES) Cellule

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Biologia

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivazione di cellule staminali embrionali umane da Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e manutenzione di cellule staminali embrionali umane

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparazione Stendere cromosomiche di cellule staminali embrionali umane per cariotipo

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelamento e scongelamento cellule staminali embrionali umane

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Trasfettando e Nucleofecting umani cellule staminali pluripotenti indotte

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

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

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...