Questo lavoro è stato supportato da un assegno di ricerca completa del California Institute per la medicina rigenerativa (RC1-00104), un Pubblico Servizio Sanitario Grant (HL085377) da NHLBI, e un dono da parte della Fondazione Pollin di HSB

Il protocollo scritto che segue si basa su parametri pubblicati segnalati dagli autori, con alcune modifiche. Il protocollo è eseguita con l&#39;approvazione del Istituzionali Animal Care UCSF e utilizzo (IACUC) e Cell Stem Oversight di ricerca (SCRO) Comitati. Cultura I. cellule staminali embrionali umane (hESC) <ol><li> HESC crescere in standard 6-bene (3,5 cm di diametro pozzi) piatti. </li><li> Almeno 12 ore prima passaging cellule, mouse fibroblasti semi embrionali (MEF; ottenuti da CF1 giorni e12.5 topi tempo-accoppiati, irradiato a 1000 Ci) alimentatore cellule su piastre rivestite con gelatina 1% (6,7). MEF dovrebbero essere placcato confluenza al 50-60%, o 4 x 10 5 cellule per 3,5 cm di diametro e (Figura 1). </li><li> Celle di passaggio, quando le colonie hanno raggiunto confluenza ~ 70% (Figura 2). </li><li> Per il passaggio, le cellule aspirare KSR terreno di coltura (6) dai pozzetti e sostituirlo con 0,5 ml di mezzo contenente Collagenasi IV KSR ad una concentrazione di 1 mg / ml. </li><li> Incubare le piastre con collagenasi IV a 37 ° C / 5% di CO 2 per 5 minuti o finché i bordi di colonie cominciano a sollevare dal piatto. </li><li> Rimuovere Collagenasi IV dal piatto, aggiungere 0,5 ml di KSR in ogni pozzetto, e manualmente raschiare le cellule per rimuovere completamente dalla piastra. </li><li> Raccogliere la sospensione cellulare da ogni pozzetto, posto in un tubo da 15 ml, e consentono alle cellule di regolare dalla forza di gravità per 5 minuti. </li><li> Eliminare il surnatante dal tubo, lasciando le cellule e medie in un volume totale di ~ 300μl. </li><li> Utilizzando un 200 ul micro-pipetta insieme a tutto volume, con delicatezza pipetta la sospensione cellulare 5-6 volte a rompere le colonie. </li><li> Portare la sospensione cellulare per un volume totale di 9 ml per pozzetto originale di celle usando KSR medio (cioè, se uno così è raccolto, il tubo deve contenere 9 ml). Integrare la media con fattore ricombinante umano fondamentale di crescita dei fibroblasti (bFGF) ad una concentrazione di 12,5 ng / ml. </li><li> Dopo aver lavato i nuovi pozzi che contiene cellule MEF (vedi punto 2) con fosfato di Dulbecco soluzione salina tamponata (DPBS) per rimuovere tutti i siero, aggiungere 3 ml di sospensione cellulare in ogni pozzetto e incubare le piastre a 37 ° C / 5% di CO 2. </li><li> Cambiamenti a medio KSR con bFGF 24 ore dopo il passaggio, e ogni 24-48 ore fino a quando le cellule sono pronte per essere diversi passaggi ancora. </li></ol> II. Preparazione di hESC pellet per l&#39;innesto <ol><li> A 24 ore prima del trapianto, aggiungere inibitore ROCK al mezzo KSR ad una concentrazione finale di 10μM per aumentare la vitalità cellulare (8). Un pozzetto di cellule a confluenza ~ 70% (5 x 10 5 cellule) creerà due palline, con due palline inserito per capsula renale. </li><li> Per preparare pellet cellulari per l&#39;innesto (9), incubare trattati con l&#39;inibitore culture ROCK con collagenasi IV per 5 minuti a 37 ° C / 5% di CO 2. </li><li> Aspirare il IV Collagenasi dal pozzo e sostituirlo con 1 DPBS ml con 10% di penicillina / streptomicina (pen / strep). </li><li> Raschiare manualmente le colonie dalla piastra, lavare il pozzo con un ulteriore 1 DPBS ml con la penna / strep, e trasferire ciascuna aliquota 1 ml di sospensione cellulare in una provetta da microcentrifuga 1,5 ml. </li><li> Spin tubi microcentrifuga brevemente a 8.000 x g, aspirare il supernatante e risospendere il pellet in DPBS 500μl con la penna / strep. </li><li> Per aiutare a legare insieme le cellule per il trapianto, aggiungere 100μl 1 mg / ml lectina Phaseolus vulgaris (PHA) in DPBS ad ogni provetta. </li><li> Incubare le cellule / sospensione PHA a temperatura ambiente per 5 minuti, poi girare per 2 minuti a 8.000 x g. Ruotare i tubi a 180 ° all&#39;interno del loro pozzi rotore e ruotare ancora per 2 minuti a 8.000 x g. </li><li> Con attenzione aspirare il sopranatante lasciando intatto il pellet. </li><li> Sloggiare i pellet cellulari dai tubi pipettando gentilmente KSR 200μl accanto al bordo del pellet fino a che non solleva lentamente. </li><li> Una volta rimosso, trasferire immediatamente il pellet a 0.4μm inserti MILLICELL collocati in pozzi cm 3.5 di diametro contenente 3 KSR ml, e incubare per 2 ore per tutta la notte a 37 ° C / 5% di CO 2. </li></ol> III. Capsula renale innesto <ol><li> Eseguire tutte le procedure animali in conformità con le linee guida istituzionali con le necessarie tecniche asettiche. </li><li> Anestetizzare topi SCID beige (CB17.Cg-Prkdc scid LYST bg / CRL), di età compresa tra 8-12 settimane, utilizzando una combinazione di O2: 3% per via inalatoria isoflurane/0.5% e tribromoethanol intraperitoneale (Avertin; 25 mg / kg, preparati al momento). Due pellet cellulari per il mouse sarà innestata mediante le tecniche descritte (10). </li><li> Dopo aver posizionato il mouse su una piastra elettrica per prevenire l&#39;ipotermia, radere l&#39;area incisione, poi disinfettare con clorexidina. Fai da 2,5 centimetri un&#39;incisione attraverso la pelle, appena fuori del centro, ma in parallelo alla colonna vertebrale. </li><li> Mantenere la zona umida con incisione sterile Salinposta, fare una piccola incisione attraverso il muscolo, appena sotto le costole e ~ 1 cm dalla colonna vertebrale. L&#39;incisione dovrebbe essere appena sufficiente per tirare il rene attraverso, ma abbastanza piccolo che il rene non scivolare indietro nella cavità addominale senza forza. </li><li> Tirare delicatamente il rene attraverso l&#39;incisione del muscolo utilizzando una pipetta Pasteur di vetro che è stato tirato in forma di un ottuso, gancio leggermente ricurva. </li><li> Bagnate il rene con DPBS sterile per evitare che la capsula si secchi. Questo può essere necessario ripetere tutta la procedura, in quanto la capsula è molto fragile. </li><li> Utilizzando Dumont pinze # 5, estrarre delicatamente la capsula fino dal rene e fare una incisione 2 millimetri con le forbici a molla Vännäs. </li><li> Con un&#39;altra pipetta di vetro che è stato tirato in un finissimo, sonda smussata, fare una piccola tasca tra la capsula e il rene. </li><li> Utilizzando un grande orifizio puntale posizionato su una pipetta di trasferimento monouso, prendere una sola cella di pellet da inserire MILLICELL. Assicurati di prendere il meno mezzi di comunicazione più lungo possibile, come supporti in eccesso possono interferire con il trasferimento a pellet. </li><li> Tenendo premuto il bordo dell&#39;incisione per creare la tasca, poggiate delicatamente il pellet accanto alla apertura e spingerlo nella tasca verso un polo del rene con la pipetta tirato. </li><li> Ripetere la procedura con un secondo pellet. Inserire questo sotto la capsula del rene stesso, ma verso il polo opposto. </li><li> Accuratamente posto la capsula posteriore verso il basso sulla pellet. Il pellet deve rimanere all&#39;interno della tasca, e non sono necessari punti di sutura per chiudere la capsula. </li><li> Spingere delicatamente il rene indietro nella cavità addominale e chiudere la parete muscolare con punti di sutura in seta 4-0 (o inferiore) con annesso reverse-taglio ago. L&#39;incisione dovrebbe essere abbastanza piccolo che solo una sutura singola è necessario. </li><li> Chiudere la pelle con una serie di 4-5 punti interrotta, utilizzando lo stesso materiale di sutura. </li><li> Prima di posizionare il mouse indietro nella sua gabbia, amministrare 0,05 mg / kg di buprenorfina (11,12) e 5 mg / kg di ketoprofene (12,13) ​​come analgesici a breve termine ea lungo termine, rispettivamente. Sostanze controllate dovrebbe essere utilizzato solo in conformità alle direttive istituzionali. </li><li> Trasferire l&#39;animale alla sua gabbia e permettono di recuperare da anestesia su una piastra elettrica. Questo dovrebbe prendere circa 20-30 minuti. </li><li> Una volta che il mouse è completamente ambulatoriale, può essere restituito alla struttura stabulazione degli animali. </li></ol> IV. Teratoma raccolta, fissazione e sezionamento <ol><li> A 8-12 settimane post-intervento chirurgico, ci dovrebbe essere un teratoma appena palpabile in prossimità del rene. Grande, pieno di liquido cisti si sviluppano spesso pure, a partire da 6-8 settimane. Questi possono richiedere un raccolto prima se causano disagi all&#39;animale. Dal teratomi crescere a ritmi più lenti rispetto cisti, però, si vuole aspettare più a lungo possibile per ottenere un teratoma di dimensioni sufficienti per l&#39;analisi (cioè, almeno 8 settimane). </li><li> Rimuovere il rene, teratoma, e qualsiasi cisti che circonda come un blocco di tessuto dalla cavità addominale utilizzando lo stesso approccio chirurgico mediante il quale il rene è stato originariamente accessibile (cfr. Sezione III, i passaggi 1-4 sopra). </li><li> Posizionare l&#39;intero blocco di tessuto asportato in una provetta contenente formalina tamponata al 10%. </li><li> Lasciare che il teratoma per fissare una notte a 4 ° C. </li><li> Il giorno seguente, togliere il teratoma dal tubo. Sezionare via qualsiasi cisti renali e per quanto possibile, senza danneggiare il teratoma stesso (Figura 3). Grandi teratomi possono essere tagliati a metà per facilitare la dissezione e incorporamento. </li><li> Elaborare il teratoma utilizzando uno standard di paraffina fissazione tecnica. Unità automatiche sono disponibili (ad esempio, Hypercenter Shandon): <table border="0"><tbody><tr><td> I. </td><td> Flex 80% di alcol </td><td> 2 ore </td></tr><tr><td> II. </td><td> Flex 80% di alcol </td><td> 1 ora </td></tr><tr><td> III. </td><td> Flex 85% di alcol </td><td> 1,5 ore </td></tr><tr><td> IV. </td><td> 95% di alcol Flex </td><td> 1 ora </td></tr><tr><td> V. </td><td> 100% Flex alcool </td><td> 1 ora </td></tr><tr><td> VI. </td><td> 100% Flex alcool </td><td> 1 ora </td></tr><tr><td> VII. </td><td> 100% Flex alcool </td><td> 1 ora </td></tr><tr><td> VIII. </td><td> Clear-Rite 3 </td><td> 1 ora </td></tr><tr><td> IX. </td><td> Clear-Rite 3 </td><td> 1 ora </td></tr><tr><td> X. </td><td> Clear-Rite 3 </td><td> 1 ora </td&gt; </tr><tr><td> XI. </td><td> Paraffina (TissuePrep) </td><td> 2 ore </td></tr><tr><td> XII. </td><td> Paraffina (TissuePrep) </td><td> 2 ore </td></tr></tbody></table></li></ol> V. immunoistochimica e analisi <ol><li> Una volta inclusi in paraffina, sezione teratoma a 5 micron fette di serie con un microtomo rotativo, e le sezioni galleggiante su vetrini. </li><li> Prima della colorazione, cuocere le diapositive per almeno un&#39;ora. </li><li> Macchiare le diapositive con ematossilina e eosina utilizzando metodi standard per identificare le strutture del tessuto rappresentante di ciascuno dei tre strati germinali embrionali (Figura 4): <table border="0"><tbody><tr><td> I. </td><td> Lavare Xilene </td><td> 3-5 minuti </td></tr><tr><td> II. </td><td> Xilene </td><td> 3-5 minuti </td></tr><tr><td> III. </td><td> 100% di alcol </td><td> 3-5 minuti </td></tr><tr><td> IV. </td><td> 100% di alcol </td><td> 3-5 minuti </td></tr><tr><td> V. </td><td> 95% di alcol </td><td> 3-5 minuti </td></tr><tr><td> VI. </td><td> 80% di alcol </td><td> 3-5 minuti </td></tr><tr><td> VII. </td><td> Acqua di lavaggio (diverse modifiche) </td><td> 2 minuti </td></tr><tr><td> VIII. </td><td> Ematossilina di Gill # 3 </td><td> 2-5 minuti </td></tr><tr><td> IX. </td><td> Acqua di lavaggio (diverse modifiche) </td><td> finché l&#39;acqua non è pulita </td></tr><tr><td> X. </td><td> Acqua di Scott (per nuclei blu) </td><td> 3 minuti o più </td></tr><tr><td> XI. </td><td> Acqua di lavaggio (diverse modifiche) </td><td> 1-2 minuti </td></tr><tr><td> XII. </td><td> Eosina Y </td><td> 1 minuto </td></tr><tr><td> XIII. </td><td> Acqua di lavaggio (diverse modifiche) </td><td> 30 secondi </td></tr><tr><td> XIV. </td><td> 80% di alcol </td><td> 1 minuto </td></tr><tr><td> XV. </td><td> 95% di alcol </td><td> 2 minuti </td></tr><tr><td> XVI. </td><td> 95% di alcol </td><td> 2 minuti </td></tr><tr><td> XVII. </td><td> 100% di alcol </td><td> 3 minuti </td></tr><tr><td> XVIII. </td><td> 100% di alcol (pulito) </td><td> 3 minuti </td></tr><tr><td> XIX. </td><td> Xilene </td><td> 2 minuti o più </td></tr><tr><td> XX. </td><td> Xilene </td><td> 2 minuti o più </td></tr></tbody></table></li><li> Montare un coprioggetti su ogni diapositiva con Permount. </li></ol> VI. Rappresentante Risultati <img src="/files/ftp_upload/2036/2036fig1.jpg" alt="Figura 1" /> Figura 1. Placcatura di fibroblasti di topo irradiati CF1 embrionale come strato alimentatore per hESC coltura indifferenziato. MEF sono placcati in confluenza ~ 50%, come mostrato qui, o 4 x 10 5 cellule per 3,5 cm di diametro e in un 6-ben piatto cultura. Bar, 100 micron. <img src="/files/ftp_upload/2036/2036fig2.jpg" alt="Figura 2" /> Figura 2. Esempio di cultura subconfluent di hESC indifferenziata su uno strato di alimentatore MEF. HESC sono coltivate su livelli alimentatore MEF fino a ~ 70% confluenti, come mostrato qui, poi diversi passaggi come descritto. Bar, 100 micron. <img src="/files/ftp_upload/2036/2036fig3.jpg" alt="Figura 3" /> Figura 3. Esempio di teratomi espiantati. Seguito espianto, il teratoma, il rene, e qualsiasi tessuto accessorio sono fissati come un blocco in formalina, poi il tessuto dei reni e accessori sono mondate accuratamente via prima inclusione in paraffina. <img src="/files/ftp_upload/2036/2036fig4.jpg" alt="Figura 4" /> Figura 4. Teratomi derivati ​​da hESC indifferenziato contenere rappresentante tessuti di tutti i tre foglietti embrionali in vivo. Un teratoma, come quello illustrato nella Figura 3, è stato sezionato e colorati con ematossilina e eosina per identificare i tessuti embrionali. hESC dare origine a tessuti derivati ​​da tutti e tre i foglietti embrionali embrionali (ectoderma (A, B), mesoderma (C), endoderma (D). (A) nascente struttura del tubo neurale. (B) epitelio squamoso primitivo. (C) Cartilagine circondato da capsula del mesenchima condensato. (D) la struttura ghiandolare intestinale. Bar, 100 micron. Le immagini riprodotte con l&#39;autorizzazione dell&#39;autore. <img src="/files/ftp_upload/2036/2036fig5.jpg" alt="Figura 5" /> Figura 5. Analisi teratoma può essere utilizzato per mappare il destino di tag, hESC indifferenziato. Come precedentemente pubblicato (9), una miscela di hESC specificamente taggati con hESC senza tag può essere utilizzato per mappare il destino delle cellule tag in un test di formazione teratoma. Come mostrato qui, hESC indifferenziato che esprime il marcatore di superficie, CD133, e contrassegnati con maggiore proteina verde fluorescente (eGFP), sono state mescolate con untagged, hESC indifferenziato. Questi sono stati usati per formare teratomi dalla capsula renale innesto. Analisi immunoistochimica di ematossilina e eosina-macchiate sezioni teratoma con anticorpi anti-eGFP dimostra il destino neuroectodermici del hESC CD133 +. Teratomi sono stati trattati come in Figura 4 e di contrasto con anticorpi anti-eGFP (marrone) per la localizzazione dei derivati ​​di cellule CD133 + GFP + all&#39;interno dei tessuti. CD133 + derivate cellule (frecce) sono stati osservati nei tessuti derivanti da ectoderma embrionale, in particolare epitelio neurale (a sinistra) e la nascente tubo neurale-come le strutture (a destra). Bar, 100 micron. Le immagini riprodotte con l&#39;autorizzazione dell&#39;autore. Gelatina <ul><li> 0,1% di gelatina suina </li><li> 99,9% DPBS <ul><li> Aggiungere la gelatina alla DPBS, incubare a 37 ° C per un&#39;ora o fino a quando tutti i gelatina va in soluzione e filtrare sterilizzare (0.22μm) prima dell&#39;uso. </li></ul></li></ul> MEF media <ul><li> 10% di siero fetale bovino (qualificata) </li><li> 90% Dulbecco Modified Medium (D-MEM) </li><li> 1x L-Glutammina </li><li> 1x Pen / Strep <ul><li> Filtro sterilizzare prima dell&#39;uso. </li></ul></li></ul> KSR (Sostituzione Knockout siero) di medie <ul><li> 20% KnockOut sostituzione siero </li><li> KnockOut 80% di D-MEM </li><li> 1x L-Glutammina </li><li> 1x Amino Acids non essenziali </li><li> 0.1 mM β-mercaptoetanolo / </li><li> 12,5 ng / ml bFGF <ul><li> Filtro sterilizzare. </li><li> Conservare a 4 ° C, ma calda a 37 ° C prima di aggiungere alle cellule. </li><li> Aggiungi bFGF immediatamente prima dell&#39;uso. </li></ul></li></ul> Collagenasi IV <ul><li> Sciogliere 1 mg / ml nel terreno di KSR mettendo a 37 ° C per 1-2 ore o fino a quando tutta la polvere entra in soluzione. </li><li> Filtro sterilizzare. </li></ul> Avertin <ul><li> 0,25 g 2,2,2-tribromoethanol </li><li> 0,25 ml di 2-metil-2-butanolo <ul><li> Incubare a 37 ° C per una notte per formare una soluzione al 100%. </li><li> Diverse ore prima dell&#39;intervento chirurgico, soluzione diluita al 2,5% di lavoro utilizzando DPBS e incubare a 37 ° C per almeno 1 ora. </li><li> Filtro sterilizzare e aliquota. </li><li> Tribromoethanol è altamente sensibile alla luce, in modo da proteggere dalla luce in tutte le fasi di preparazione e di utilizzo. </li><li> Preparare non più di 12-24 ore prima dell&#39;uso. </li></ul></li></ul> Buprenorfina <ul><li> 1 mg / ml di soluzione concentrata in metanolo <ul><li> Diluire a 0,015 mg / ml di soluzione sterile di lavoro con H 2 O. </li><li> Conservare a -20 ° C fino a 1 mese. </li></ul></li></ul> Ketoprofene <ul><li> 0,5 mg / ml di soluzione di lavoro nel DPBS <ul><li> Aggiungi DPBS, incubare una notte a 37 ° C per 1-2 ore o fino a quando tutta la polvere entra in soluzione. </li><li> Filtro sterilizzare e conservare a 4 ° C. </li></ul></li></ul>

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C., Ducrot, R., Pasquet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+pharmacological+and+toxicological+studies+on+ketoprofen.">Some pharmacological and toxicological studies on ketoprofen.</a> Rheumatol Rehabil. , (1976).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo adattato un saggio altamente efficiente per la formazione teratoma ai fini della mappatura del destino di differenziare hESC in un ambiente nel vivo. Un piccolo numero di hESC appositamente selezionato viene mescolato con indifferenziato hESC wild-type e la lectina Phaseolus vulgaris a formare un pellet di cellule. Questo si innesta sotto la capsula del rene in un mouse immunodeficienti. Il destino del hESC selezionati in origine può essere determinata mediante immunoistochimica (Figura 5), come riportato in precedenza (9). Questo metodo fornisce un prezioso strumento per l&#39;individuazione e la generazione di tessuti specifici reagenti per la terapia cellulare.

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2-Methyl-2-butanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>240486</td>
<td>Avertin</td>
</tr>
<tr>
<td>2,2,2-Tribromoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T48402</td>
<td>Avertin</td>
</tr>
<tr>
<td>4-0 silk sutures</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>641G</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>233FB</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B7536</td>
<td>&nbsp;</td>
<tr>
<td>Clear-Rite 3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046-341</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C5138</td>
<td>&nbsp;</td>
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>Dupont #5 forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>500233</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>Eosin Y</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>23-245-658</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (Qualified)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Flex alcohol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046344</td>
<td>&nbsp;</td>
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<td>Gill&#x2019;s Hematoxylin #3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-050-204</td>
<td>&nbsp;</td>
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<td>Hemostat, straight</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501241</td>
<td>General surgery</td>
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<td>Iris forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>15914</td>
<td>General surgery</td>
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<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>K2012</td>
<td>&nbsp;</td>
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<td>KnockOut D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10829</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25020-081</td>
<td>&nbsp;</td>
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<td>MILLICELL inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM01250</td>
<td>&nbsp;</td>
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<td>Nonessential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>Operating scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501754</td>
<td>General surgery</td>
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<tr>
<td>Paraffin (TissuePrep)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>8002-74-02</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Permount</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SP15-100</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L1668</td>
<td>&nbsp;</td>
<tr>
<td>Porcine gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G6144</td>
<td>&nbsp;</td>
<tr>
<td>ROCK inhibitor</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>Y-27632</td>
<td>&nbsp;</td>
<tr>
<td>SCID beige mice</td>
<td>&nbsp;</td>
<td>Charles River </td>
<td>250</td>
<td>&nbsp;</td>
<tr>
<td>Scott&#x2019;s Wash</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>6697-32</td>
<td>Ricca Chemicals</td>
</tr>
<tr>
<td>Vannas spring scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>14003</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>		</tbody>
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fate mapping of human embryonic stem cells by teratoma formation

Cellule staminali embrionali umane (hESC) hanno una capacità illimitata di auto-rinnovamento, e la capacità di differenziarsi in cellule derivate da tutti e tre i foglietti embrionali embrionali (1). Regia di hESC differenziazione in tipi cellulari specifici ha suscitato molto interesse nel campo della medicina rigenerativa (ad esempio, (2-5)) e metodi per la determinazione della<em&gt; In vivo</emDestino&gt; di hESC selezionati o manipolati sono essenziali per questa impresa. Abbiamo adattato un saggio altamente efficiente formazione teratoma per questo scopo. Un piccolo numero di hESC appositamente selezionato viene mescolato con indifferenziato hESC wild-type e la lectina Phaseolus vulgaris a formare un pellet di cellule. Questo si innesta sotto la capsula del rene in un mouse immunodeficienti. Da un minimo di 2,5 x 10<sup&gt; 5</supHESC&gt; sono necessari per formare un 16 centimetri<sup&gt; 3</supTeratoma&gt; entro 8-12 settimane. Il destino del hESC selezionati in origine può essere determinata mediante immunoistochimica. Questo metodo fornisce un prezioso strumento per la caratterizzazione di tessuto-specifici reagenti per la terapia cellulare.

Watch this Scientific Journal Video about Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation at JoVE.com

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Regia di hESC differenziazione in cellule specifiche, ha generato molto interesse nella medicina rigenerativa. Forniamo un conciso, passo-passo il protocollo per la determinazione della<em&gt; In vivo</emDestino&gt; di hESC selezionato che fornisce un valido strumento per la caratterizzazione di tessuto-specifici reagenti per la terapia cellulare.

Mappatura destino di cellule staminali embrionali umane per la formazione teratoma

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

fate-mapping-of-human-embryonic-stem-cells-by-teratoma-formation

Research

JoVE Journal

Biology

17.1K Views.  University of California San Francisco. Regia di hESC differenziazione in cellule specifiche, ha generato molto interesse nella medicina rigenerativa. Forniamo un conciso, passo-passo il protocollo per la determinazione della In vivo di hESC selezionato che fornisce un valido strumento per la caratterizzazione di tessuto-specifici reagenti per la terapia cellulare.

Video: Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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

Ricerca

Biologia

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondaggio per mitocondriale complessa attività di cellule staminali embrionali umane

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Etichettatura in vitro di cellule staminali embrionali umane per la Risonanza Magnetica

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

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 ...

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 ...