Questo manoscritto è dedicato alla memoria del Dr. Wang Wenlan scomparso il 26 maggio 2011. Ringraziamo il dottor Douglas A. Kerr generosamente per fornire i HBG3 cellule MES usati in questo studio. Questo lavoro è stato finanziato da Nemours, una borsa di studio (2 RR016472-10) nell&#39;ambito del programma INBRE del Centro nazionale per le risorse di ricerca (NCRR), e un premio COBRE borsa di studio del NCRR (5 RR020173 P20-05) per sostenere il Centro per La ricerca pediatrica presso l&#39;Alfred I. duPont Hospital for Children, Wilmington, Delaware, USA.

1. Fase 1: Mouse Embryonic Stem (MES) Cultura Cell (Timing: 3 giorni) 1. Placcatura fibroblasti primari embrionali di topo (PMEF) <ol><li> Cappotto quattro da 100 mm di coltura dei tessuti piatti con gelatina di adesione PMEF. Aggiungere 8 ml di 0,1% soluzione di gelatina (StemCell Technologies) per ogni piatto e incubare per 30 min a temperatura ambiente. </li><li> Togliere l&#39;eccesso di gelatina da piatti. Non sciacquare i piatti. </li><li> Diluire una fiala di Mitomicina C-inattivato PMEF (Millipore) che contiene ca. 5 x 10 6 cellule in 40 ml di PMEF supporti (vedi ricetta in tabella 1) e la piastra su quattro piatti tessuto 100-mm di coltura. Il PMEF fornire uno strato di alimentazione per le cellule MES. </li><li> PMEF devono essere seminati almeno un giorno prima di aggiungere colture cellulari MES. PMEF può essere usato fino a 1 settimana dopo la placcatura. </li></ol> 2. Placcatura MES Celle Per monitorare l&#39;efficienza della differenziazione cellulare MES in motoneuroni, Abbiamo utilizzato HBG3, una linea di cellule ES derivata da un topo transgenico che esprime eGFP sotto il controllo del promotore motore HB9 neurone specifico. <ol><li> Sbrinamento 1 flacone di HBG3 cellule MES (circa 1 x 10 7, ha contribuito dal dottor Douglas Kerr, Johns Hopkins University) in un bagno di acqua a 37 ° C. Aggiungere 5 ml di ES supporti (vedi ricetta in tabella 1) in una provetta da 15 ml e centrifugare le cellule a 800 rpm per 5 min. </li><li> Aspirare il surnatante e risospendere le cellule in MES 20 ml di supporto ES con fresco aggiunto fattore inibitorio della leucemia (LIF) ad una concentrazione finale di 10 ng / ml. NOTA: LIF deve essere aggiunto il giorno di utilizzo ed è necessario per mantenere le cellule MES uno stato indifferenziato. </li><li> Rimuovere 10 ml supporti PMEF dal PMEF in ogni piatto e aggiungere 10 ml di sospensione HBG3 cellule ES ad ogni piatto. </li><li> 24 h dopo la placcatura, cellule MES formano colonie piccole di dimensioni simili a colonia indicata dalla freccia in figura 1B. 72 h dopo la placcatura, colonies aumento delle dimensioni, molti essendo circa 100 micron di diametro (figura 1B, indicato dalle frecce). Queste cellule sono pronte per la differenziazione. I terreni devono essere sostituiti ogni giorno per mantenere la proliferazione delle cellule. </li></ol> 2. Fase 2: induzione neurale (Timing: 2 giorni) Per indurre la differenziazione di cellule SEM nel motoneuroni, cellule MES devono essere separati da PMEF e coltivato in un ambiente sospensione. Gelatina fiaschi rivestiti sono usati per separare PMEF dalle cellule MES. <ol><li> Quando le colonie MES sono pronti per l&#39;induzione neurale (definito come giorno differenziazione 0), mantello 2 fiasche T150 con gelatina 0,1% (18 ml / beuta) per 30 min a temperatura ambiente e quindi rimuovere gelatina eccesso e lavare con PBS 1X tre volte. NOTA: Ogni 100-mm cultura piatto sarà necessario uno T150-gelatina pallone rivestito per separare PMEF. </li><li> Rimuovere con attenzione dei media per aspirazione, facendo attenzione a non rimuovere le colonie vagamente collegati. Aggiungere 10 ml di PBS brevemente e quindi rimuovere per aspirazione per completare la fase di lavaggio. </li><li> Per separare le cellule MES di PMEF, aggiungere 0,25% di tripsina / EDTA (3 ml / piatto, StemCell Technologies) e incubare per 3-5 minuti a 37 ° C finché le cellule staminali e PMEF staccarsi dalla piastra. Aggiungere 15 ml supporti ES freschi senza LIF e le cellule pipettare su e giù per rompere colonie (circa 15-20 volte). Quindi, aggiungere a una gelatina rivestita 0,1% T150 pallone e incubare per 30 min a 37 ° C. Dopo l&#39;incubazione, PMEF dovrebbe attaccarsi alla superficie gelificato mentre le cellule staminali embrionali dovrebbe essere galleggiare. Raccogliere le cellule galleggianti MES in una provetta da 50 ml. </li><li> Centrifugare a 800 rpm e risospendere le cellule in 10 ml di Medium differenziamento neurale (vedi ricetta in Tabella 1). </li><li> Contare le cellule utilizzando un emocitometro e poi piastra circa 2 x 10 6 cellule MES su un piatto di 100-mm coltura batterica o sospensione. Queste piastre permettono la crescita di colture in sospensione e promuovere la formazione di corpi embrionali (EBS). </li><li> On differentiazione 1 giorno, le cellule MES formare piccoli aggregati fluttuanti (EBS) che sono visibili al microscopio ottico. Qualsiasi PMEF riporto allegare al piatto. Trasferire le cellule in sospensione e media in un tubo da 15 ml e centrifugare a bassa velocità (500 rpm) per 3 min. Aspirare i media con attenzione, aggiungere 10 ml di fresca Medio differenziamento neurale al pellet EBS, e poi le cellule piastra in un nuovo piatto batterica. Oltre a rifornire i mezzi di comunicazione, questo passaggio rimuove qualsiasi PMEF che riporto dalla fase precedente. </li><li> Il giorno 2 differenziazione, EBS sono pronti per essere indotto in neuroni motori. </li></ol> 3. Fase 3: Motor Neuron Specification (Timing: 5 giorni) <ol><li> Il giorno 2 differenziazione, agitare il piatto della cultura e trasferire la media e EBs in una provetta da 15 ml. Lasciate EBs risolvere per gravità (~ 10 min) oppure mediante centrifugazione a bassa velocità (500 rpm) per 3 min. </li><li> Aspirare il terreno con cura e risospendere EBs in 10 ml di differenziazione Motor Neuron (MND), medio (vedi ricetta in <strong&gt; Tabella 1). Piastra EBs in un nuovo piatto batterica e modificare i supporti giorno per 5 giorni. </li><li> Il giorno 7 differenziazione, EBs dovrebbe essere di circa 150 a 200 micron in diametro e dovrebbe esprimere segnali forti GFP (figura 1C). Si tratta di EB sono pronti per essere dissociati e placcato su poly-DL-ornithine/laminin piatti rivestiti o coprioggetti. </li></ol> 4. Fase 4: Preparazione del Poly-DL-ornithine/laminin Coprivetrini rivestite (Timing: 2 giorni) Due giorni prima dissociare EBS (cioè il giorno differenziazione 5), preparare coprioggetti poly-DL-ornithine/laminin-coated. <ol><li> Luogo 12 mm coprioggetto rotondi (Warner Instruments) sul fondo di una piastra da 24 pozzetti. Sciogliere 25 mg di poli-DL-ornitina (Sigma-Aldrich) in 25 ml sterile, acqua bidistillata (d 2 H 2 O) per ottenere una soluzione 10X fotografia (1 mg / ml). Al momento dell&#39;uso, una diluizione 1/10 di poli-DL-ornitina con d 2 H 2 O per ottenere concentrazione di 100 ug / ml. Aggiungere 0,5 ml a ciascun pozzetto della piastra da 24 pozzetti ed incubare a 4 ° C per il pernottamento. </li><li> Il giorno successivo (differenziazione giorno 6), aspirare poli-DL-ornitina e lasciare che le piastre di copertura e scivola aria secca in un cappuccio coltura tissutale. Risciacquare nei pozzetti della piastra con D 2 H 2 O tre volte. Lasciare asciugare all&#39;aria per un&#39;altra ora. </li><li> Sciogliere 1 mg del mouse laminina (Millipore) in 5 ml di freddo-freddo 1X PBS per rendere disponibile una soluzione 100x (200 mg / ml). Al momento dell&#39;uso, fare diluizione 1/100 in ghiacciato-freddo 1X PBS (2 mg laminina / ml). Aggiungere 0,5 ml / pozzetto in una piastra da 24 pozzetti e incubare a 4 ° C per il pernottamento. Prima che le cellule di semina, laminina in eccesso deve essere rimosso e pozzi risciacquati con PBS 1X due volte. Copri possono essere memorizzati in MND mezzo (0,5 ml / pozzetto). </li></ol> 5. Fase 5: Allungamento Axon (Timing: 2 giorni) <ol><li> Il giorno 7 differenziazione, raccogliere EBs in un tubo da 15 ml e permettono di risolvere EB (circa 3 minuti). Aspirare media e ri-suspend EBs in PBS. Lasciare EBS per insediarsi e poi aspirare PBS. Ripetere questa operazione 3 volte. Aggiungere 1 ml di Accumax (Millipore) per l&#39;EBS, mescolare delicatamente e incubare per 5 min a temperatura ambiente. Poi aspirare Accumax eccesso che copre il EBS. </li><li> Per dissociare EBS, aggiungere 3 ml di terreno MND. Pipettare su e giù per 20 volte usando un ml 1 micropipetta Eppendorf (blu punta) per interrompere EBS. Incubare per 2 min a temperatura ambiente. Trasferire sospensione cellulare in un tubo mm 12x75 con cellule filtro tappo (BD Falcon) per ottenere una sospensione di cellule singole. Ripetere questo passaggio dissociazione di ciuffi e le cellule rimanenti trattenuti dal filtro, in modo da arricchire la resa di singole cellule. La sospensione finale singola cellula sarà 6 ml. </li><li> Mescolare delicatamente questa sospensione singola cella e contare le cellule utilizzando un emocitometro. Diluire le cellule nel mezzo MND (supplementato con 10 ng / ml di ciascun BDNF, GDNF, CNTF, e NT-3) ad una densità di 2x10 5 cellule per ml. Aggiungere sospensione cellulare (0,5 ml / pozzetto) a 24-pozzetti contenenti pocoprioggetti ly-DL-ornithine/laminin rivestite (precedentemente preparato come descritto sopra). Le cellule differenziate si estendono neuriti a lungo dopo 2 giorni di coltura (Figura 1D). </li></ol> 6. Risultati rappresentativi Figura 1A mostra il profilo del protocollo. Nel passaggio 1, le cellule sono coltivate in MES PMEF e integrato con LIF per prevenire differenziazione spontanea. In generale, indifferenziate colonie MES sono rotondi e compatti, e sono chiaramente definiti i bordi. Le colonie non sono in contatto tra loro. Tipicamente, le cellule MES dovrebbe essere diviso in un rapporto 1:4 ogni 2 ~ 3 giorni. Tuttavia, ciascuna linea singola cellula crescerà ad un tasso differente e il rapporto di divisione devono essere determinate empiricamente. Le frecce in figura 1B indicano l&#39;aspetto tipico di cellule MES dopo coltura su uno strato alimentatore PMEF per 3 giorni. Queste colonie sono di grandi dimensioni (50-100 micron di diametro), ma mantengono ancora orlo tondo e definitos. In questa fase le colonie sono pronti per la differenziazione o sottocultura. Una punta di freccia in Figura 1B mostra una piccola colonia MES che solitamente si trova 24 h dopo la placcatura. Quattro giorni dopo la placcatura, le cellule diventano MES invaso. Essi mostrano un aspetto appiattito e la perdita di confini definiti, che indica che stanno cominciando a differenziare. Tali cellule non sono ottimali per la differenziazione nelle cellule neuronali. Per differenziare le cellule MES in neuroni motori, le cellule MES bisogno di essere coltivata in condizioni di sospensione per permettere la formazione EB. Nel passaggio 2, cellule ES sono trasferite su una superficie di coltura senza uno strato alimentatore per promuovere la formazione EB. In questo passo, vengono incubate in mezzo differenziamento neurale supplementato con Noggin, bFGF, e FGF-8 per 2 giorni a cellule dirette ad lignaggio neurale. Piccola EBS può essere osservata al microscopio a giorno 1 di differenziazione. Dovrebbero essere galleggiare nel mezzo. Riporto PMEF si possono trovare anche al giorno 1di differenziazione e devono essere rimossi. Queste cellule batteriche aderiscono ai piatti in modo vengono eliminati quando EBS sono diversi passaggi ad un nuovo piatto batterica. Così, al giorno 2 di differenziazione, PMEF pochi o nessun va visto nel piatto di coltura. Al punto 3, ha continuato il trasferimento di EBS per nuovi piatti devono assicurare la rimozione di qualsiasi PMEF rimanente. EBS sono coltivate nella differenziazione dei motoneuroni (MND), i media integrati con RA e SAG per 5 giorni di differenziare le cellule in neuroni motori. Durante la coltura, EB continuano a crescere in dimensione e può essere visto ad occhio nudo al giorno differenziazione 3 ~ 4. Figura 1C mostra l&#39;aspetto microscopico di differenziazione EBs il giorno 7. A differenza delle cellule indifferenziate, EBS mostrano una forte fluorescenza a causa di espressione di GFP. Si tratta di EBS sono differenziati in modo ottimale e sono pronti per la dissociazione. Citometria a flusso dimostrato che il 51% ± 0,8% delle cellule dal dissociato EBs era differenziate in cellule esprimenti GFP 5. Al punto 5, della cultura della thEse motoneuroni in presenza di GDNF, CNTF, BDNF, NT3 e risultati in estensione di lunghe proiezioni assonale. figura 1D mostra la comparsa di cellule differenziate 2 giorni dopo placcatura di dissociata EBs. Nota neuriti lunghi si estendono dai corpi cellulari delle cellule placcati. Colorazione immunofluorescente mostra che le cellule GFP + esprimono il pan-marcatore neuronale (neurofilamento-catena media) e due motori marcatori neuronali specifici, (Islet-1 e colina acetiltransferasi, Figura 2). <img alt="Figura 1" src="/files/ftp_upload/3813/3813fig1.jpg" /> Figura 1. Differenziazione di cellule SEM nel motoneuroni (immagine modificata da Wu et al. 5). A) Schema di protocollo di differenziazione da cellule MES per i motoneuroni. B) le cellule indifferenziate MES formano colonie rotonde sulla parte superiore di uno strato di fibroblasti alimentatore. Hanno debole espressione GFP. C) le cellule MES sono state separate da strati feeder e culturosso a basso attaccamento piatti a formare EBS. Durante i primi due giorni del processo di differenziazione, le cellule sono state esposte a 50 ng / Noggin ml, 20 ng / ml bFGF, e 20 ng / ml FGF-8. Successivamente, sono state indotte a differenziarsi con 1 pM acido retinoico e 1 pM sonic SAG agonista hedgehog per 5 giorni. Differenziate le cellule MES ha espresso una forte fluorescenza GFP. D) Dopo 7 giorni di differenziazione, EBS erano dissociate e placcato su poly-DL-ornithine/laminin piastre rivestite. Le cellule differenziate esteso lunghi processi neurali dopo 2 giorni di coltura. Scala bar = 200 micron. MN = motoneurone. B, C e D sono immagini in campo chiaro e B &#39;, C&#39; e D &#39;sono corrispondenti immagini a fluorescenza. <img alt="Figura 2" src="/files/ftp_upload/3813/3813fig2.jpg" /> Figura 2. Caratterizzazione di MES derivati ​​da cellule neuroni motori. Immunofluorescenza per catena neurofilamenti medio (NF-M, rosso), chat (rosso), e Islet-1 (rossa) è stata coincidente con GFP (green) exprESSIONE. DAPI (blu) è stato utilizzato per identificare i nuclei. Scala bar = 50 micron.

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La qualità di cellule MES è il parametro più critico per la produzione efficiente di neuroni motori. MES cellule devono essere coltivate in PMEF per prevenire differenziazione spontanea. L&#39;aggiunta di LIF aiuta a mantenere le cellule MES uno stato indifferenziato. Come cellule MES dividere ogni 12-15 ore, il terreno di coltura si esaurisce rapidamente e deve essere sostituito ogni giorno. Efficiente attivazione del pathway Shh è un parametro critico necessario per indurre motore neurone specifica. Proteina Shh (R &amp; D System), e Shh agonisti segnalazione come agonista Hh HhAg1.3 (Curis, Inc.), purmorphamine (Calbiochem) e SAG (EMD Chemicals) sono stati utilizzati per promuovere la formazione di neuroni motori 1-3 , 10,11. Abbiamo trovato SAG essere una molecola più efficace purmorphamine nella differenziazione delle cellule verso un fenotipo neuronale motore 5. 1-2,5 pM purmorphamine con pM RA 1 non ha una efficienza di differenziazione di oltre il 20%. Tuttavianelle nostre mani una combinazione di 1 pM di RA e 1 pM di SAG ha un&#39;efficienza differenziazione di circa il 25% in procedure senza una fase di induzione neurale. Per migliorare ulteriormente l&#39;efficienza differenziazione, si aggiunge un giorno 2-passo neurale induzione prima motore passo-passo neurone specifica. Questo approccio sfrutta il fatto che sia Noggin e segnalazione FGF sono essenziali per la fase iniziale di induzione neurale, quando le cellule embrionali primo luogo inserire il lignaggio neurale 6-9. Oltre a induzione neurale, FGF segnalazione mantiene culture, come le cellule precursori neurali. Sovraespressione di Fgf8 nel mesencefalo in via di sviluppo porta ad una drammatica espansione della popolazione precursore neurale nella zona ventricolare 12. Una combinazione di FGF e Noggin agisce sinergicamente per indurre la formazione di tessuto neurale attraverso la promozione di una identità neurale posteriore 9. Così il giorno 2-passo di induzione neurale è progettato per dirigere le cellule MES versoil lignaggio neurale prima dell&#39;inizio del processo di specificazione dei motoneuroni. Il protocollo qui descritto è una modifica e il miglioramento del protocollo originale stabilito descritto in precedenza 1. Anche se la timeline di generare neuroni motori è la stessa, abbiamo fatto una serie di modifiche per snellire la procedura e migliorare la resa dei motoneuroni. Aggiungendo un passo neurale induzione al protocollo differenziazione, siamo in grado di aumentare l&#39;efficienza differenziazione dal 25% al ​​50%. Il nostro protocollo prevede un approccio efficace per arricchire motoneuroni derivati ​​dalle cellule MES. Motoneuroni non può essere ottenuto da pazienti SMA e la cattiva salute dei motoneuroni primari da topi SMA esclude l&#39;isolamento di cellule sufficienti quantità, qualità e purezza di effettuare analisi quantitative di proteine ​​colpiti in questa malattia. Utilizzando questo protocollo cellule MES, siamo stati in grado di ottenere una popolazione di cellule del motoneurone quan sufficienteidentità e la purezza di uno studio proteomica per identificare i meccanismi molecolari coinvolti nella atrofia muscolare spinale 5.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Concentrazione finale </td></tr><tr><td> PMEF </td><td> Millipore </td><td> PMEF-H </td><td> NA </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> PMEF media </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965-118 </td><td> NA </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16140-071 </td><td> 10% </td></tr><tr><td> L-glutammina </td><td> Invitrogen </td><td> 25030-081 </td><td> 1% </td></tr><tr><td> Penicillina / streptomicina </td><td> Invitrogen </td><td> 15240-063 </td><td> 1% </td></tr><tr><td></td><td></td&gt; <td></td><td></td></tr><tr><td> MES media </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell Technologies </td><td> 36250 </td><td> NA </td></tr><tr><td> ES Cell-Qualified FBS </td><td> Invitrogen </td><td> 16141-079 </td><td> 15% </td></tr><tr><td> -I Glutamax </td><td> Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> Aminoacidi non essenziali </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Nucleosidi </td><td> Millipore </td><td> ES-008-D </td><td> 1% </td></tr><tr><td> β-mercaptoetanolo </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> Penicillina / streptomicina </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> LIF </td><td> Millipore </td><td&gt; LIF2010 </td><td> 10 ng / ml </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Differenziazione medio Neural </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell Technologies </td><td> 36250 </td><td> NA </td></tr><tr><td> ES Cult FBS </td><td> StemCell Technologies </td><td> 06905 </td><td> 15% </td></tr><tr><td> Aminoacidi non essenziali </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Mono-tio glicerolo </td><td> Sigma-Aldrich </td><td> M-6145 </td><td> 1mM </td></tr><tr><td> Boccaletto </td><td> Invitrogen </td><td> PHC1506 </td><td> 50 ng / ml </td></tr><tr><td> FGF-8 </td><td> Invitrogen </td><td> PHG0274 </td><td> 20 ng / ml </td></tr><tr><td> bFGF </td><td> Invitrogen </td><td> PHG0024 </td><td> 20 ng / ml </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> MND medio (differenziazione) </td><td></td><td></td><td></td></tr><tr><td> ES-Cult Basal Medium-A </td><td> StemCell Technologies </td><td> 5801 </td><td> NA </td></tr><tr><td> Knockout sostituzione siero </td><td> Invitrogen </td><td> 10828-028 </td><td> 10% </td></tr><tr><td> N-2 integratore </td><td> Invitrogen </td><td> 17502-048 </td><td> 1% </td></tr><tr><td> ITS Supplement-B </td><td> StemCell Technologies </td><td> 07155 </td><td> 1% </td></tr><tr><td> Acido ascorbico </td><td> StemCell Technologies </td><td> 07157 </td><td> 1% </td></tr><tr><td> Penicillina / streptomicina </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> -I Glutamax </td><td>Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> D-glucosio </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% in d 2 H 2 O </td></tr><tr><td> Fibronectina </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 pg / ml </td></tr><tr><td> Eparina </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 ug / ml in d 2 H 2 O </td></tr><tr><td> β-mercaptoetanolo </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> Acido Retinoico </td><td> Sigma-Aldrich </td><td> R-2625 </td><td> 1 pM </td></tr><tr><td> SAG </td><td> EMD Chemicals </td><td> 566660 </td><td> 1 pM </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> MND medio (Motor Neuron cultura) </td><td></td><td></td><td></td></tr><tr><td> ES-Cult Basal Medium-A </td><td> StemCell Technologies </td><td> 5801 </td><td> NA </td></tr><tr><td> Knockout sostituzione siero </td><td> Invitrogen </td><td> 10828-028 </td><td> 10% </td></tr><tr><td> N-2 integratore </td><td> Invitrogen </td><td> 17502-048 </td><td> 1% </td></tr><tr><td> ITS Supplement-B </td><td> StemCell Technologies </td><td> 07155 </td><td> 1% </td></tr><tr><td> Acido ascorbico </td><td> StemCell Technologies </td><td> 07157 </td><td> 1% </td></tr><tr><td> Penicillina / streptomicina </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> -I Glutamax </td><td> Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> D-glucosio </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% in d 2 H 2 O </td></tr><tr><td> Fibronectina </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 pg / ml </td></tr><tr><td> Eparina </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 ug / ml in d 2 H 2 O </td></tr><tr><td> β-mercaptoetanolo </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> BDNF </td><td> R &amp; D Systems </td><td> 248-BD-005/CF </td><td> 10 ng / ml </td></tr><tr><td> CNTF </td><td> R &amp; D Systems </td><td> 257-NY-010/CF </td><td> 10 ng / ml </td></tr><tr><td> GDNF </td><td> R &amp; D Systems </td><td> 212-GD-010/CF </td><td> 10 ng / ml </td></tr><tr><td> NT-3 </td><td> R &amp; D Systems </td><td> 267-N3-005/CF </td><td> 10 ng / ml </td></tr></tbody></table> NA = Non applicabile. Tabella 1. PMEF e dei media per la cultura o la differenziazione delle cellule MES. <table border="1"><tbody><tr><td> &lt;&gt; Nome forte del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Concentrazione finale </td></tr><tr><td> 0,1% gelatina </td><td> StemCell Technologies </td><td> 07903 </td><td> NA </td></tr><tr><td> Poly-DL-ornitina </td><td> Sigma-Aldrich </td><td> P0421 </td><td> 0,1 mg / ml in d 2 H 2 O </td></tr><tr><td> Mouse laminina </td><td> Millipore </td><td> CC095 </td><td> 2 ug / ml in PBS </td></tr><tr><td> 0,25% di tripsina / EDTA </td><td> StemCell Technologies </td><td> 07901 </td><td> NA </td></tr><tr><td> Accumax </td><td> Millipore </td><td> SCR006 </td><td> NA </td></tr></tbody></table> NA = Non applicabile. Tabella 2. Reagenti per il rivestimento e la dissociazione.

efficient differentiation of mouse embryonic stem cells into motor neurons

Differenziazione diretta delle staminali embrionali (ES) in cellule funzionali motoneuroni rappresenta una risorsa promettente per studiare i meccanismi della malattia, per lo screening di nuovi composti farmacologici, e per sviluppare nuove terapie per malattie del motoneurone come l&#39;atrofia muscolare spinale (SMA) e la sclerosi laterale amiotrofica ( ALS). Molti attuali protocolli utilizzano una combinazione di acido retinoico (RA) e sonic hedgehog (Shh) per differenziare staminali embrionali di topo (MES), le cellule in neuroni motori 1-4. Tuttavia, l&#39;efficienza differenziazione delle cellule SEM nel motore neuroni ha soltanto incontrato con discreto successo. Abbiamo sviluppato un protocollo due fasi differenziazione 5 che migliora significativamente l&#39;efficienza di differenziazione rispetto a protocolli attualmente applicate. Il primo passo è di migliorare il processo neuralization aggiungendo Noggin e fattori di crescita dei fibroblasti (FGF). Noggin è una proteina morfogenetica dell&#39;osso (BMP), antagonista ed è implicato in uno induzione neuraleecondo al modello predefinito di neurogenesi e risulta nella formazione di anteriore patterning neurale 6. FGF segnalazione agisce sinergicamente con Noggin nell&#39;indurre la formazione di tessuto neurale attraverso la promozione di una identità neurale posteriore 7-9. In questa fase, le cellule sono state MES innescato con Noggin, bFGF, e FGF-8 per due giorni per promuovere la differenziazione verso lignaggi neurali. Il secondo passo è quello di indurre le specifiche dei neuroni motori. Noggin / FGFs esposti MES cellule sono state incubate con RA e un agonista Shh, agonista Smoothened (SAG), per altri 5 giorni per facilitare la generazione dei neuroni motori. Per monitorare la differenziazione di MES in neuroni motori, abbiamo utilizzato una linea di cellule ES derivata da un topo transgenico che esprime eGFP sotto il controllo del promotore motore neurone specifico HB9 1. Usando questo protocollo robusto, abbiamo raggiunto 51 ± 0,8% di efficienza differenziazione (n = 3; p &lt;0,01, t di Student-test) 5. I risultati di immunofluorescenzacolorazione ha dimostrato che le cellule GFP + esprimere i marcatori dei motoneuroni specifici, Islet-1 e colina acetiltransferasi (Chat). I nostri due fasi protocollo di differenziazione offre un modo efficace per differenziare le cellule MES in neuroni motori spinali.

Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Abbiamo sviluppato un nuovo protocollo per migliorare l&#39;efficienza del differenziamento in vitro delle cellule staminali embrionali di topo in neuroni motori. Le cellule staminali differenziate acquisito motoneuroni presenta come evidenziato dalla espressione di marcatori neuronali e motorie dei neuroni che utilizzano tecniche immunoistochimiche.

Differenziazione efficiente di cellule staminali embrionali di topo in neuroni motori

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

efficient-differentiation-mouse-embryonic-stem-cells-into-motor

Nanyang Technological University

Research

JoVE Journal

Biology

22.2K Views.  Alfred I. duPont Hospital for Children. Abbiamo sviluppato un nuovo protocollo per migliorare l'efficienza del differenziamento in vitro delle cellule staminali embrionali di topo in neuroni motori. Le cellule staminali differenziate acquisito motoneuroni presenta come evidenziato dalla espressione di marcatori neuronali e motorie dei neuroni che utilizzano tecniche immunoistochimiche.

Video: Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

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 ...

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 Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

La differenziazione in vitro di staminali embrionali di topo (MES) Cellule con il metodo goccia

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...