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 neur...

<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 ...

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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

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 ...

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Biologia

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 ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Generazione rapida e efficiente di neuroni da cellule staminali umane pluripotenti in un formato piatto Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Differenziazione del neonato mouse pelle cellule staminali derivate in cellule germinali-simili<em&gt; In vitro</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

L&#39;infezione retrovirale di Murine staminali embrionali cellulari derivate Cellule embrionali di corpo per l&#39;analisi di differenziazione emopoietiche

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...