Questo lavoro è stato sostenuto dalla National Natural Science Foundation of China (n. 31501099) e dalla Mezza età e Dai Giovani del Dipartimento dell'Istruzione della Provincia di Hubei, Cina (n. trimestre 20191104). E ringraziamo il professor Wensheng Deng alla Wuhan University of Science and Technology per aver fornito le linee di cellule staminali embrionali del topo A2lox.

1. Coltura delle cellule staminali embrionali del topo
<ol><li>Preparare piatti o piatti di coltura cellulare ricoperti di gelatina 0,1%.<ol>
<li>Aggiungere 2 mL di gelatina sterilizzata allo 0,1% (0,1% w/v in acqua) a piatti di coltura cellulare da 60 mm. Dondolare delicatamente per garantire un rivestimento uniforme delle stoviglie di coltura cellulare.</li>
		<li>Mettere i piatti in un incubatore di CO2 al 5% a 37 °C e lasciare il rivestimento per 1 h.</li>
		<li>Rimuovere la soluzione di gelatina allo 0,1% prima di seminare le cellule. NOTA: Dopo aver rimosso la gelatina, non è necessario asciugare o lavare i piatti rivestiti.</li>
	</ol>
</li>
	<li>Coltura delle cellule staminali embrionali del topo (A2lox e 129)<ol>
<li>Incubare le cellule mESC (A2lox e 129) nell'incubatore di coltura cellulare 0,1% rivestito di gelatina da 60 mm in un mezzo di crescita mESC rispettivamente a 37 °C in un incubatore di CO2 al 5%. Il mezzo di crescita mESC è costituito per l'85% da DMEM/F12, per il 15% da sostituti del siero knock-out (KSR), per 0,1 mM β-mercaptoetanolo (2ME), 2 mM glutaMAX, 1% amminoacido non essenziale (NEAA), 1% penicillina/streptomicina (P/S), 1000 U/mL fattore inibitorio della leucemia (LIF), 10 nM CHIR-99021 (inibitore GSK-3) e 0,33 nM PD0325901 (inibitore MEK). ATTENZIONE: β-mercaptoetanolo è infiammabile e presenta tossicità per inalazione. Tenere lontano dalle fonti di fuoco e indossare una maschera per evitare l'inalazione durante l'uso.</li>
		<li>Cambia quotidianamente il mezzo di crescita mESC per una migliore crescita di A2lox e 129.</li>
		<li>Quando le cellule raggiungono l'80% di confluenza, rimuovere il mezzo e aggiungere 1 mL dello 0,1% di tripside al piatto. Dondolare delicatamente per 30 s per garantire una copertura uniforme della tripside su tutte le cellule.</li>
		<li>Lasciare le celle per circa 1 minuto per tripinaree quindi rimuovere la tripina utilizzando pipetta da 1 mL.</li>
		<li>Aggiungere più volte 2 mL di mezzo di crescita mESC al piatto, pipetta su e giù più volte per effettuare una sospensione a cella singola.</li>
		<li>Contare la densità delle cellule nella sospensione nel modo più accurato possibile usando l'emocitometro.</li>
		<li>Dividere le celle in 7 gruppi e indurre la differenziazione utilizzando protocolli diversi indicati nella tabella 1.</li>
	</ol>
</li>
</ol>2. Differenziazione dai MESC ai PNG (fase I)
<ol><li>Preparare 0,1% piatti di coltura cellulare rivestiti di gelatina o coverlips.<ol>
<li>Prima dell'uso, preparare 0,1% di gelatina rivestita con 6 piastre o coverlips come nel passaggio 1.1.</li>
	</ol></li>
	<li>Differenziazione della fase I mediante protocollo n. 1(tabella 1)<ol>
<li>Seme circa 2 x 104 mESC in 2 mL di mezzo di differenziazione basale I per pozzo nelle piastre a 6 pozzetti rivestite con gelatina allo 0,1%. Controllare la densità cellulare al microscopio.</li>
		<li>Incubare le cellule a 37 °C in un incubatore di CO2 al 5% per 6 ore per consentire l'attacco.</li>
		<li>Dopo l'attacco, eserti dalle cellule dall'incubatrice e lavare le cellule due volte con 2 mL di PBS.</li>
		<li>Aggiungere 2 mL di mezzo di differenziazione basale I (Tabella 1) a ciascun pozzo e rimettere le cellule nell'incubatrice.</li>
		<li>Lasciare le cellule per la differenziazione per 8 giorni. Sostituire il mezzo di differenziazione basale I ogni 2 giorni.</li>
	</ol></li>
	<li>Differenziazione della fase I mediante protocollo n. 2(tabella 1)<ol>
<li>Aggiungere 1,5 x 106 mESC in un piatto batterico non adeso in 10 mL di mezzo di differenziazione basale I per consentire la formazione del corpo embrionale a 37 °C in un incubatore di CO2 al 5%.</li>
		<li>Dopo 2 giorni, trasferire gli aggregati cellulari in tubi di centrifuga da 15 ml e lasciarli depositare per gravità.</li>
		<li>Rimuovere il supernatante e aggiungere 10 mL di mezzo di differenziazione basale fresco I per risospendire i corpi embrioidi. Ripiantarli in un nuovo piatto batterico non adeso e consentire la differenziazione per altri 2 giorni.</li>
		<li>Controllare la formazione di corpi embrioidi al microscopio (Figura 2A).</li>
		<li>Raccogliere corpi embrio embrioni come descritto nei passaggi 2.3.2-2.3.3. Semina circa 50 corpi embriobi in 2 mL di mezzo di differenziazione basale I per pozzo su piastre a 6 porci ricoperte di gelatina allo 0,1%.</li>
		<li>Preparare 1 mM di materiale RA tutto trans (in DMSO) e conservare lontano dalla luce in un congelatore a -80 °C dopo il sub-imballaggio. NOTA: RA è instabile e si dovrebbe prestare attenzione a tenerlo lontano dalla luce e ridurre il contatto con l'aria durante la preparazione del materiale RA.</li>
		<li>Per l'induzione RA, aggiungere 2 μL di materiale RA in ogni pozzo per effettuare una concentrazione finale di 1 μM.</li>
		<li>Posizionare la piastra nell'incubatore di CO2 al 5% a 37 °C e differenziare per altri 4 giorni.</li>
		<li>Cambiare l'intero mezzo di differenziazione basale I (con 1 μM RA) ogni 2 giorni.</li>
	</ol></li>
	<li>Differenziazione della fase I mediante protocollo n. 3(tabella 1)<ol>
<li>Piantare 1,5 x 106 mESC in un piatto batterico non adeso in 10 mL di mezzo di differenziazione basale I. Lasciare per 2 giorni per la formazione del corpo embrionale a 37 °C in un incubatore di CO2 al 5%.</li>
		<li>Controllare la formazione di corpi embrioidi al microscopio (Figura 2B).</li>
		<li>Trasferire gli aggregati cellulari in tubi di centrifuga da 15 ml e lasciarli depositare per gravità.</li>
		<li>Rimuovere con cura il supernatante e aggiungere 10 mL di mezzo di differenziazione basale fresco I per risospendirli.</li>
		<li>Seminare circa 50 corpi embrioidi in 2 mL di mezzo di differenziazione basale I per pozzo su piastre a 6 porci ricoperte di gelatina allo 0,1%.</li>
		<li>Per l'induzione RA, aggiungere 2 μL di materiale RA in ogni pozzo per effettuare una concentrazione finale di 1 μM.</li>
		<li>Posizionare la piastra nell'incubatore di CO2 al 5% a 37 °C e differenziare per altri 6 giorni. Cambiare l'intero mezzo di differenziazione basale I (con 1 μM RA) ogni 2 giorni.</li>
	</ol></li>
	<li>Differenziazione della fase I mediante protocollo n. 4(tabella 1)<ol>
<li>Seme circa 2 x 104 mESC entro 2 mL dal mezzo di differenziazione basale I per pozzo sulle piastre a 6 pozzetti rivestite con gelatina allo 0,1%. Posizionare la piastra nell'incubatore di CO2 al 5% a 37 °C per consentire l'attacco per 6 ore.</li>
		<li>Dopo l'attacco, lavare le celle due volte con 2 mL di PBS. Aggiungere 2 mL di mezzo di differenziazione basale I ad ogni pozzo e consentire la differenziazione per 4 giorni nell'incubatore di CO2 al 5% a 37 °C.</li>
		<li>Per l'induzione RA, aggiungere 2 μL di materiale RA completamente trans in ogni pozzo (la concentrazione di lavoro è di 1 μM) per indurre la differenziazione per altri 4 giorni.</li>
		<li>Nell'intero processo, sostituire l'intero mezzo ogni 2 giorni.</li>
	</ol></li>
	<li>Differenziazione della fase I mediante protocollo n. 5(tabella 1)<ol>
<li>Seme circa 2 x 104 mESC entro 2 mL dal mezzo di differenziazione basale I per pozzo sulle piastre a 6 pozzetti rivestite con gelatina allo 0,1%. Posizionare la piastra nell'incubatore di CO2 al 5% a 37 °C per consentire l'attacco per 6 ore.</li>
		<li>Lavare le cellule due volte con 2 mL di PBS. Aggiungere 2 mL di mezzo di differenziazione basale I ad ogni pozzo e consentire la differenziazione per 2 giorni nell'incubatore di CO2 al 5% a 37 °C.</li>
		<li>Per la successiva induzione RA, aggiungere 2 μL di materiale RA in ogni pozzo per effettuare una concentrazione finale di 1 μM. Posizionare la piastra nell'incubatore di CO2 del 5% a 37 °C per indurre la differenziazione per altri 6 giorni.</li>
		<li>Nell'intero processo, sostituire l'intero mezzo ogni 2 giorni.</li>
	</ol></li>
	<li>Differenziazione della fase I mediante protocollo n. 6(tabella 1)<ol>
<li>Piantare 1,5 x 106 mESC in una piastra batterica non adesiva in 10 mL di N2B27 medium II(tabella 1)per consentire la formazione di corpi embrioidi.</li>
		<li>Il 2° giorno, raccogliere gli aggregati cellulari come descritto nei passaggi 2.3.2-2.3.3 e resuspend i corpi embrioidi utilizzando 10 mL di N2B27 medio II fresco.</li>
		<li>Ripiantarli in un nuovo piatto batterico non adeso e consentire la differenziazione per altri 2 giorni nell'incubatore di CO2 al 5% a 37 °C. Controllare la formazione di corpi embrioidi al microscopio.</li>
		<li>Il 4,raccogliere corpi embrionale. Semina circa 50 corpi embriombrioidi per pozzo su piatti a 6 po 'rivestiti di gelatina 0,1% con 2 mL di N2B27 medium II.</li>
		<li>Aggiungere 2 μL di materiale RA completamente trans in ogni pozzo e indurre la differenziazione per altri 4 giorni. Sostituire l'intero mezzo (N2B27 medium II con 1 μM RA) ogni due giorni.</li>
	</ol></li>
	<li>Differenziazione della fase I mediante protocollo n. 7(tabella 1)<ol>
<li>Seme circa 2 x 104 mESC entro 2 mL dal mezzo di differenziazione basale I per pozzo sulle piastre a 6 pozzetti rivestite con gelatina allo 0,1%. Posizionare la piastra nell'incubatore di CO2 al 5% a 37 °C per consentire l'attacco per 6 ore.</li>
		<li>Lavare le celle due volte con PBS. Quindi, aggiungere 2 mL di N2B27 medium II ad ogni pozzo e consentire la differenziazione per 8 giorni a 37 °C in un incubatore di CO2 al 5%.</li>
		<li>Cambiare l'intero N2B27 medium II ogni 2 giorni.</li>
	</ol></li>
</ol>3. Osservazione della morfologia cellulare
<ol><li>Controllare lo stato di differenziazione dei suddetti 7 gruppi al giorno sotto un microscopio a contrasto di fase invertito.</li>
	<li>Seleziona casualmente almeno 12 campi e scatta foto per registrare i cambiamenti morfologici di ogni gruppo su D8.</li>
</ol>4. Colorazione immunofluorescenza
<ol><li>Preparazione del campione: Semi mESC su coverlips rivestiti di gelatina allo 0,1% e consentire la differenziazione per 8 giorni utilizzando i protocolli menzionati nella fase 2.</li>
	<li>Risciacquo: L'8 ° giorno, prelevare i campioni dall'incubatore e rimuovere il mezzo di differenziazione per aspirazione. Risciacquare delicatamente le cellule una volta con 1 mL di PBS per 5 minuti.</li>
	<li>Fissazione: Aggiungere 1 mL di paraformaldeide al 4% a ciascun campione e fissare le cellule per 20 minuti a temperatura ambiente (RT).</li>
	<li>Risciacquo: Dopo la fissazione, sciacquare delicatamente le cellule con 1 mL di PBS 3 volte, per 5 minuti ciascuna.</li>
	<li>Permeabilizzazione: Aggiungere 1 mL dello 0,2% di TritonX-100 in PBS a ciascun campione e lasciare per 8 minuti a RT.</li>
	<li>Risciacquo: Dopo la permeabilità, sciacquare delicatamente le cellule con 1 mL di PBS 3 volte, per 5 minuti ciascuna.</li>
	<li>Blocco: Aggiungere 1 mL di siero di capra al 10% in PBS a ciascun campione e incubare a RT per 1 h per bloccare eventuali interazioni non specifiche.</li>
	<li>Incubazione con anticorpo primario<ol>
<li>Diluire l'anticorpo anti-nestina con un rapporto di 1:100 usando il 5% di siero di capra in PBS.</li>
		<li>Applicare 500 μL di anticorpi diluiti su diversi campioni e incubare durante la notte a 4 °C.</li>
	</ol>
</li>
	<li>Risciacquare: Rimuovere l'anticorpo e risciacquare delicatamente i campioni con 1 mL di PBS 3 volte per 8 minuti ciascuno.</li>
	<li>Incubazione con anticorpo secondario<ol>
<li>Diluire l'IgG anti-topo di capra etichettato Alexa Fluor 488 con un rapporto di 1:500 usando il 5% di siero di capra in PBS.</li>
		<li>Applicare 500 μL di anticorpo diluito su diversi campioni e incubare al buio per 2 ore a RT. NOTA: Dopo aver applicato anticorpi secondari fluorescenti, eseguire tutti i passaggi successivi al buio per evitare la tempra a fluorescenza.</li>
	</ol>
</li>
	<li>Risciacquare: Rimuovere l'anticorpo secondario e risciacquare delicatamente i campioni con 1 mL di PBS 3 volte per 8 minuti ciascuno.</li>
	<li>Colorazione e montaggio nucleare<ol>
<li>Posizionare una goccia di supporto di montaggio DAPI sul microslide pulito.</li>
		<li>Eslevare con cura i campioni dalle piastre e posizionare il campione sopra il supporto di montaggio DAPI con la cella a faccia in giù. Lasciare al buio per 5 minuti a RT.</li>
		<li>Rimuovere il mezzo di montaggio DAPI in eccesso con carta assorbente.</li>
	</ol>
</li>
	<li>Osservazione della microscopia a fluorescenza<ol>
<li>Posizionare i campioni sotto la microscopia a fluorescenza e rilevare il segnale per DAPI e Alexa Fluor 488 utilizzando filtri adeguati.</li>
		<li>Scegli in modo uniforme e casuale 10-15 campi visivi diversi per ogni campione e registra le immagini con una fotocamera CCD.</li>
	</ol>
</li>
</ol>5. Differenziazione dai PNG ai neuroni (Fase II)
<ol><li>Preparare i derivati mESC nell'ambito della differenziazione di fase I utilizzando il protocollo 3 (8giorni, tabella 1 ) come descritto nella fase 2.4, che ha la massima efficienza di differenziazione (cfr. figura 3). NOTA: Dopo la differenziazione della fase I, il controllo di qualità deve essere effettuato utilizzando l'osservazione della morfologia cellulare e il saggio di immunofluorescenza sopra menzionato, per garantire un NPC sano e ad alto rendimento.</li>
	<li>Seme circa 5 x 105 derivati mESC entro 2 mL dal mezzo di differenziazione basale I per pozzo sulle piastre a 6 porci rivestite con gelatina allo 0,1%. Dividi casualmente le derivate mESC in 3 gruppi, rispettivamente come protocollo di fase II 1, protocollo 2 e protocollo 3.</li>
	<li>Posizionare la piastra nell'incubatore di CO2 al 5% a 37 °C per consentire l'attacco per 6 ore. Lavarli due volte con 2 mL di PBS.</li>
	<li>Aggiungere 2 mL di mezzo di differenziazione basale I, N2B27 medio I e N2B27 medio II (tabella 2), rispettivamente, a ciascun pozzo dei gruppi di cui sopra.</li>
	<li>Posizionare le piastre nell'incubatore e lasciare differenziare per altri 10 giorni. Cambiare il mezzo corrispondente ogni 2 giorni.</li>
	<li>Controllare lo stato di differenziazione e registrare i cambiamenti morfologici come menzionato nel passaggio 3.</li>
	<li>Il giorno 18, valutare la generazione di neuroni (β-Tubulina III positivo) e determinare l'efficienza di differenziazione dei 3 protocolli utilizzando nel passaggio 4.</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid-induced+upregulation+of+miR-219+promotes+the+differentiation+of+embryonic+stem+cells+into+neural+cells.">Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells.</a> Cell Death Disease. 8, 2953 (2017).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+regulates+germ+cell+differentiation+in+mouse+embryonic+stem+cells+through+a+Smad-dependent+pathway.">Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway.</a> Biochemical and Biophysical Research Communications. 418 (3), 571-577 (2012).</li><li>Nakayama, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+neuronal+induction+of+the+P19+embryonic+carcinoma+cell+line.">A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line.</a> Journal of Neuroscience Methods. 227, 100-106 (2014).</li><li>Bibel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+into+a+defined+neuronal+lineage.">Differentiation of mouse embryonic stem cells into a defined neuronal lineage.</a> Nature neuroscience. 7 (9), 1003-1009 (2004).</li><li>Coyle, D. E., Li, J., Baccei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Differentiation+of+Retinoic+Acid-Induced+Human+Pluripotent+Embryonic+Carcinoma+Stem+Cell+Neurons.">Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons.</a> PLoS ONE. 6 (1), 16174 (2011).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Developmental Biology. 275, 124-142 (2004).</li></ol>

Gli autori non hanno nulla da rivelare.

Nel presente studio, abbiamo stabilito un metodo semplice ed efficace per la differenziazione neuronale dagli mESC, con materiali a basso costo e facilmente ostruibili. In questo metodo, 2 giorni di formazione del corpo embrionale seguiti da 6 giorni di induzione ra possono promuovere efficacemente la differenziazione degli MESC in NPC (protocollo di fase I 3). Per la differenziazione di fase II, N2B27 medium II (Phase II-protocol 3) induce più efficacemente la differenziazione dai PNG ai neuroni. Per garantire il succe...

Le cellule staminali embrionali (ESC) sono pluripotenti e possono differenziarsi in cellule precursori neurali (NPC) e successivamente in neuroni in determinate condizioni1. La neurogenesi basata su ESC fornisce la migliore piattaforma per imitare la neurogenesi, fungendo così da strumento utile per gli studi di biologia dello sviluppo e potenzialmente aiutare nella medicinarigenerativa 2,3. Negli ultimi decenni, sono state riportate molte strategie per indurre la neurogenesi embrionale, come il metodo transgenico4, utilizzandopiccolemolecole 5,utilizzando un microambiente a matrice 3D6e la tecnica di co-coltura7. Tuttavia, la maggior parte di questi protocolli sono condizioni limitate o difficili da usare, quindi non sono adatti per l'uso nella maggior parte dei laboratori.
Per trovare un metodo facile da usare e a basso costo per ottenere un'efficiente differenziazione neurale dagli MESC, qui è stata utilizzata una strategia di screening combinatorio. Come descritto nella figura 1, l'intero processo di neurogenesi embrionale è stato diviso in 2 fasi. La fase I si riferisce al processo di differenziazione dagli mESC ai PNG, e la fase II si riferisce alla successiva differenziazione dai PNG ai neuroni. Sulla base dei principi di facile funzionamento, basso costo, materiali facilmente disponibili e alta efficienza di differenziazione, sono stati scelti sette protocolli nella fase I e tre protocolli nella fase II in base al tradizionale sistema di coltura monostrato aderente o al sistema di formazione del corpo embrionale8,9. L'efficienza di differenziazione dei protocolli in entrambe le fasi è stata valutata utilizzando l'osservazione della morfologia cellulare e il test di immunofluorescenza. Combinando il protocollo più efficiente di ogni fase, abbiamo stabilito il metodo ottimizzato per la differenziazione neurale dagli mESC.

<table>
	<tbody>
		<tr>
			<td>Anti-Nestin antibody [Rat-401]</td>
			<td>Abcam</td>
			<td>Ab11306</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III antibody produced in rabbit</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-Labeled Goat Anti-Mouse IgG</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>stored at -20 &#176;C, and protect from light</td>
		</tr>
		<tr>
			<td>CHIR-99021 (CT99021)</td>
			<td>Selleck</td>
			<td>S1263</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-Labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0516</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>DME/F-12 1:1 (1x)</td>
			<td>HyClone</td>
			<td>SH30023.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Gibco</td>
			<td>CM0635B</td>
			<td>stored at room temperature</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Immunol Staining Primary Antibody dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor human</td>
			<td>Sigma</td>
			<td>L5283</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI - Aqueous, Fluoroshield</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson</td>
			<td>005-000-121</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nonadhesive bacterial dish</td>
			<td>Corning</td>
			<td>3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin Solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PD0325901(Mirdametinib)</td>
			<td>Selleck</td>
			<td>S1036</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>stored at -80 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Strain 129 Mouse Embryonic Stem Cells</td>
			<td>Cyagen</td>
			<td>MUAES-01001</td>
			<td>Maintained in feeder-free culture system</td>
		</tr>
		<tr>
			<td>Stem-Cellbanker (DMSO free)</td>
			<td>ZENOAQ</td>
			<td>stem cellbanker DMSO free</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1X) Solution</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>6 - well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>NUNC</td>
			<td>339650</td>
			<td></td>
		</tr>
	</tbody>
</table>

neuronal differentiation from mouse embryonic stem cells in vitro

La differenziazione neurale delle cellule staminali embrionali del topo (mESC) è un potenziale strumento per chiarire i meccanismi chiave coinvolti nella neurogenesi e potenzialmente aiutare nella medicina rigenerativa. Qui, abbiamo stabilito un metodo efficiente e a basso costo per la differenziazione neuronale dagli mESC invitro, utilizzando la strategia dello screening combinatorio. Nelle condizioni qui definite, la formazione del corpo embrionale di 2 giorni + il protocollo di induzione dell'acido retinoico di 6 giorni consente una differenziazione rapida ed efficiente dagli MESC alle cellule precursori neurali (NPC), come visto dalla formazione di derivati A2lox e 129 ben impilati e simili a neurite che sono positivi alla Nestin. Lo stato sano dei corpi embrioidi e il momento in cui viene applicato l'acido retinoico (RA), così come le concentrazioni di RA, sono fondamentali nel processo. Nella successiva differenziazione dai NPC ai neuroni, N2B27 medium II (integrato da mezzo neurobasale) potrebbe supportare meglio il mantenimento e la maturazione a lungo termine delle cellule neuronali. Il metodo presentato è altamente efficienza, a basso costo e facile da usare e può essere un potente strumento per la neurobiologia e la ricerca sulla biologia dello sviluppo.

La formazione del corpo embrionale di 2 giorni + l'induzione ra di 6 giorni funziona meglio per dirigere la differenziazione degli MESC in NPC (Fase I). Per determinare il protocollo ottimale che promuove meglio la differenziazione degli MESC in NPC (fase I), sono stati testati 7 protocolli sia su A2lox che su 129 mESC(tabella 1)e lo stato di differenziazione di ciascun gruppo è stato monitorato utilizzando il microscopio a luce. Come mostrato nella figura 3A, la maggior pa...

Watch this Scientific Journal Video about Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro at JoVE.com

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Qui, abbiamo stabilito un metodo a basso costo e facile da usare che indirizza una differenziazione rapida ed efficiente dalle cellule staminali embrionali ai neuroni. Questo metodo è adatto per la popolarità tra i laboratori e può essere uno strumento utile per la ricerca neurologica.

Differenziazione neuronale dalle cellule staminali embrionali del topo in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

neuronal-differentiation-from-mouse-embryonic-stem-cells-in-vitro

Research

JoVE Journal

Neuroscience

8.7K Views.  Wuhan Center for Disease Control and Prevention. Qui, abbiamo stabilito un metodo a basso costo e facile da usare che indirizza una differenziazione rapida ed efficiente dalle cellule staminali embrionali ai neuroni. Questo metodo è adatto per la popolarità tra i laboratori e può essere uno strumento utile per la ricerca neurologica.

Video: Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. 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 tumorigenicity7-10. 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 problematic11-13. 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 molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivazione efficiente di progenitori neuronali umane e Neuroni pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Ricerca

Neuroscienze

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Culture Slice organotipica del mesencefalo ventrale embrionali: un sistema per studiare dopaminergici sviluppo neuronale In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Regia neuroni dopaminergici differenziazione da cellule staminali pluripotenti umane

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Differenziazione delle cellule staminali embrionali di topo in precursori Interneurone corticale

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Isolamento e coltura di cellule staminali neurali embrionali del Mouse

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

Preparazione della fetta di cuneo in vitro per imitare la connettività del circuito neuronale in vivo

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Modellazione in vitro per malattie neurologiche utilizzando la conversione diretta dai fibroblasti alle cellule progenitrici neuronali e la differenziazione in astrociti

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imaging del calcio nell'oliva inferiore del topo

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Cellule simili alla microglia umana: differenziazione da cellule staminali pluripotenti indotte e saggio di fagocitosi di cellule vive in vitro utilizzando sinaptosomi umani

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Methods Article

Differenziazione neuronale dalle cellule staminali embrionali del topo in vitro