Ringraziamo Denis Flipo per consigli elaborazione delle immagini e analisi, e David W. Silversides nel cui laboratorio è stata generata la linea di topi Gata4p [5kb]-GFP. La ricerca in laboratorio Pilon è finanziato dal CIHR, NSERC, FRQS e FRQNT.

Dichiarazione etica Gli esperimenti che coinvolgono i topi sono stati eseguiti a seguito Canadian Consiglio di linee guida per la cura degli animali per la cura e la manipolazione di animali utilizzati nella ricerca medica. Protocolli che comportano la manipolazione di animali sono stati approvati dal comitato etico istituzionale dell&#39;Università del Quebec a Montreal (Comité de Protection des institutionnel Animaux, il numero di riferimento 0512-R3-650-0513). 1. Preparazione di Collagene Gel Lavorare in modo sterile, sotto una cappa di coltura tissutale. <ol><li> Preparare completa 5x DMEM (mezzo essenziale modificata di Eagle), compresi gli antibiotici standard. Sciogliere 3,37 g di polvere DMEM e 0,925 g di NaHCO3 in 20 ml di acqua. Sterilizzare passando attraverso un filtro di 0,22 micron. Aggiungere 2,5 ml di sterile 100x penicillina / streptomicina e 25 ml di siero fetale bovino sterile calore-inattivato. Conservare a 4 ° C. </li><li> Sul ghiaccio, mescolare 800 mldi soluzione di collagene I (3,77 mg / ml in 0,02 N di acido acetico, sterilizzata per filtrazione), 600 ml di DMEM completo 5x e 17 ml di 1 N NaOH. Diluire con acqua sterile fino ad un volume finale di 3 ml. Includere i fattori di crescita rilevanti nel mix. Usiamo tipicamente GDNF a 10 ng / ml per stimolare la migrazione enterico NCC. </li><li> Deposito di circa 480 microlitri in ciascun pozzetto di una singola riga in una piastra da 24 pozzetti. Evitare le bolle. Le righe rimanenti possono essere utilizzati per verificare l&#39;effetto di altri fattori di crescita sulla migrazione cellulare. </li><li> Sia il collagene polimerizzare almeno 1 ora in un incubatore sterile a 37 ° C. </li></ol> 2. Dissezione degli animali 16 <ol><li> Impostare accoppiamenti e controllare i tappi vaginali la mattina successiva. Giorno E0.5 essendo mezzogiorno del giorno la spina vaginale si trova, isolare la femmina e attendere 12 giorni fino E12.5. </li><li> Anestetizzare topi incinta con isoflurano e eutanasia da CO 2 inalazione. </li><li> Spruzzare il mouse con etanolo al 70%. Sollevare le s addomekin e aprire la cavità addominale con le forbici dissezione. </li><li> Rimuovere l&#39;utero in una piastra di Petri di vetro riempita con PBS freddo (soluzione salina tamponata con fosfato). Tagliare l&#39;utero trasversalmente tra i singoli gonfiori deciduum per isolare ogni sito di impianto dell&#39;embrione. </li><li> Lavorare su ciascun sito separatamente impianto in un altro Petri piatto di vetro riempito con ghiaccio-freddo PBS. Sotto un microscopio da dissezione, utilizzare una pinza sottile per rimuovere lo strato muscolare dell&#39;utero. </li><li> Aprire il sacco vitellino viscerale e amnion per rivelare l&#39;embrione. Fare attenzione quando recidere i vasi sanguigni che unisce l&#39;embrione alla placenta / viscerale sacco vitellino, in quanto si intrecciano con l&#39;intestino in via di sviluppo. </li><li> Tagliare la testa dell&#39;embrione al collo. </li><li> Inserire una pinza chiuse nella cavità addominale di un embrione, appena sopra il fegato di colore rosso scuro e lasciare le pinze aperto di sé (interrompere l&#39;applicazione di pressione) per fare un&#39;apertura trasversale nella cavità addominale. Tirare le pinze aperte versol&#39;estremità posteriore dell&#39;embrione per aprire completamente l&#39;addome. </li><li> Afferra il tessuto connettivo dietro il fegato e tirare le budella dell&#39;addome, facendo attenzione a non rompere l&#39;intestino (il colon è collegato al ano). </li><li> Tagliare il colon per liberare le viscere dal resto dell&#39;embrione. Il taglio può essere effettuato in qualsiasi posizione lungo il colon. Prenota la porzione di coda per più tardi. </li><li> Giro fuori il tessuto connettivo dal cieco, poi il resto degli intestini. Fare attenzione a non ferire l&#39;intestino, mentre farlo. </li><li> Tagliare il fegato e stomaco (all&#39;estremità rostrale del piccolo intestino), così come le mesonefro e creste genitali se alcuni sono presenti. </li><li> Isolare l&#39;intestino tenue. Anche in questo caso, fare attenzione a non stroncare l&#39;intestino. Da ora in poi, l&#39;orientamento rosrrocaudal del tessuto intestinale può essere rintracciato tramite la curvatura tagliente presente sulla fine rostrale. Lasciare il piccolo intestino in PBS a temperatura ambiente per il minor tempo possibile prima emBiancheria da letto (vedi punto 3.3). </li><li> Infine, registrare il numero di somiti coda per ogni embrione per determinare con precisione lo stadio di sviluppo embrionale. </li></ol> 3. Sezionamento dei embrionali Intestino <ol><li> Prima di procedere con dissezioni embrione, sciogliere 1,5 g di agarosio in 100 ml di PBS (tampone fosfato salino) e mantenerlo a 50 ° C. </li><li> Versare agarosio fuso in uno stampo embedding (ad esempio un chiuso 2 ml provetta che è stato tagliato longitudinalmente per asportare circa 1/4 della parete del tubo). Lasciare che il agarosio raffreddare a circa 42-45 ° C, dovrebbe essere solo leggermente caldo al tatto. </li><li> Incorporare l&#39;intestino embrionale poco prima delle solidifica agarosio (questo può essere valutata con una pinza punte e si verifica intorno a 36-38 ° C). Tenendo l&#39;intestino con una pinza per la fine rostrale piegata, tirare molto lentamente attraverso l&#39;agarosio lungo la lunghezza dello stampo. Ciò contribuisce a mantenere l&#39;intestino diritto mentre i set di agarosio. Rilasciare il temettere appena comincia a resistere spostato. Tenere traccia dell&#39;orientamento rostrale-caudale dell&#39;intestino. </li><li> Mettete lo stampo in frigorifero 2-3 minuti, al fine di garantire l&#39;agarosio si è completamente impostato. </li><li> Estrarre il agarosio dallo stampo (facendola scorrere fuori dal tubo Eppendorf aperto). Con una lama, estrarre l&#39;agarosio eccesso ad entrambe le estremità, tagli perpendicolari per l&#39;intestino. </li><li> Incollare la fine rostrale dell&#39;intestino / agarosio bloccare giù sul palco di metallo di un microtomo con lama vibrante. Tagliare il agarosio eccesso sui lati del blocco dell&#39;intestino / agarosio. </li><li> Montare la fase metallica sulla camera microtomo vibrante. Se necessario, regolare l&#39;angolo della fase così l&#39;intestino è più verticale possibile (quindi perpendicolare alla lama). Coprire il campione con PBS freddo. Portare la lama microtomo fino a pochi millimetri sotto la superficie buffer. </li><li> Rendere 200 micron vibratome tagli trasversali dell&#39;intestino caudale più a piccolo, assicurando che l&#39;each fetta agarosio contiene una sezione intestinale completa. </li></ol> 4. Cultura intestinale espianti <ol><li> Depositare delicatamente il fresco tagliato a fette intestino / agarosio piatto sul gel di collagene con una pinza, ponendo una fetta verso la metà di ogni bene. </li><li> Incubare 3 giorni a 37 ° C, in un umido 5% CO 2 atmosfera, per consentire la migrazione di NCC fuori l&#39;espianto. </li><li> Prendete le fette di intestino / agarosio il gel di collagene molto delicatamente con una pinza. Fate attenzione a non danneggiare il gel sotto. </li><li> Evitare di toccare il gel di collagene direttamente durante le successive fasi di incubazione e lavaggio, al fine di assicurare che il modello migrazione cellulare non è disturbato. Fissare con 500 ml di 4% PFA (paraformaldeide) (in PBS) per pozzetto 1 ora a temperatura ambiente. </li><li> Sostituire il fissativo con 500 ml di DAPI (4 &#39;,6-diamidino-2-fenilindolo) Soluzione (5 pg / ml in PBS) per pozzetto ed incubare 10 min a temperatura ambiente. </li><li> Lavare ogni pozzetto 3xcon 500 microlitri di PBS per 5 min. </li><li> Fotografare le cellule fluorescenti (canali GFP e DAPI) incorporati nel gel di collagene in ogni pozzetto. </li></ol> 5. Image Analysis Abbiamo fatto ampio uso di ImageJ 17 per elaborare e quantificare le immagini generate dopo coltura espianto. <ol><li> Inizia l&#39;immagine di un vetrino micrometrico allo stesso ingrandimento come le fotografie di cellule fluorescenti. Misurare la lunghezza di un micron in pixel (utilizzando lo strumento linea retta). </li><li> Impostare la scala (Analyze / Set Scala, il numero di ingresso di pixel / micron). </li><li> Per ogni fotografia fluorescenza GFP, modificare il formato dell&#39;immagine a 8-bit in scala di grigi (Image/Type/8-bit). </li><li> Regolare l&#39;intensità del segnale (Foto / Regola / Luminosità / Contrasto). </li><li> Sottrarre il rumore di fondo, se necessario (Process / Sottrarre sfondo, regolare il raggio della sfera di rotolamento). </li><li> Impostare una soglia per evidenziare cellule e grumi di cellule (Immagine / Regola / Threshold), iln applicare uno spartiacque per dividere i cespi (Process / Binario / Watershed). </li><li> Analizzare le particelle di specificare regioni di interesse (ROI) e generare statistiche numero di cellulare (Analizza / Analizza particelle; dimensione minima set di escludere pixel rimanenti). </li><li> Impostare le opzioni di misura per includere il diametro di Feret (Analyze / Set Misure). </li><li> Gruppo ROI e misurare nel suo complesso per determinare il diametro della Feret, un&#39;indicazione di spread cella (Analisi / Strumenti / ROI Gestore / info / O, quindi il ROI Gestore / Aggiungi, e il ROI Gestore / Measure). </li></ol>

<ol>
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Mostriamo come il nostro ex vivo espianto cultura tecnica può essere utilizzata per quantificare con precisione enterico migrazione potenziale NCC in presenza di GDNF. Tale quantificazione precisa è notevolmente semplificata ricorrendo a 200 micron di spessore sezioni vibratome budello invece di grandi pezzi di dimensioni approssimative, come descritto in precedenza 11,12,15. In effetti, questo ci permette di lavorare con un numero ragionevole di cellule in un ambiente altamente riproducibile. Da n...

Cellule della cresta neurale (NCC) sono un tipo di cellula transitoria unica di vertebrati che si forma molti derivati ​​durante lo sviluppo embrionale. Questa popolazione di cellule si pone al confine della placca neurale, adiacente al ectoderma non-neurale 1. Durante neurulazione, piegatura dei luoghi placca neurale NCC lungo il bordo dorsale del tubo neurale formatura. NCC poi subire una transizione epitelio-mesenchimale, segregazione e la migrazione dal tubo neurale. NCC colonizzare varie strutture embrionali compresi il tratto digestivo dove formano l&#39;intero sistema nervoso enterico (ENS), una rete interconnessa di gangli nervosi incorporato nella parete intestinale. Come recentemente rivisto 2,3, molti geni sono stati coinvolti nello sviluppo di questa intricata struttura. La maggior parte della ENS è derivato da una piccola piscina di NCC provenienti dal tubo neurale vagale (ossia circa il limite prospettico hindbrain / midollo spinale) 4.Questi progenitori neurali raggiungono il foregut intorno al giorno embrionale (e) 9.0 nei topi e poi migrano caudale all&#39;interno del mesenchima intestino fino a circa e15.0 a colonizzare l&#39;intero intestino embrionali. Un sottoinsieme minore di progenitori neurali colon è fornito anche da sacrale NCC, che invadono l&#39;intestino posteriore in direzione opposta fino al cieco 4. Sia vagale e sacrale NCC richiedere più migrazione, proliferazione, sopravvivenza e spunti-differenziazione promuovere per garantire la formazione completa della ENS. A questo proposito, i modelli animali - topi geneticamente modificati, in particolare - sono state fondamentali per l&#39;identificazione dei vari ligandi extracellulari essenziali: GDNF (glial cellule fattore neurotrofico derivato), dell&#39;endotelina-3, neurotrofina-3, (proteine ​​morfogenetiche dell&#39;osso) BMP, Netrin , così come Sonic e indiano Hedgehog (Shh e Ihh) 5-10. Di questi, GDNF segnalazione attraverso il RET recettore transmembrana tirosin chinasi (Risistemato durante la transfezione), è riconosciuto come esimoe percorso più critico per l&#39;attrazione e la guida di NCC da e all&#39;interno dell&#39;intestino embrionale. GDNF è secreto dal mesenchima intestino e forma un gradiente rosrrocaudal controllato spatiotemporally che è direttamente chemoattractive a enterico NCC, che esprimono RET 11,12. Fra le altre funzioni, l&#39;ENS regola il movimento all&#39;interno del tubo digerente attraverso la sua interazione con la muscolatura liscia della parete intestinale. Assenza di gangli nervosi nella regione terminale dei risultati intestinali nella malattia di Hirschsprung: contrazione tonica del segmento interessato conduce al blocco, l&#39;accumulo a monte del materiale digerito e massiccia distensione dell&#39;intestino e dell&#39;addome. La malattia di Hirschsprung si verifica circa uno su 5.000 nati vivi. Il modello migratorio rostro-caudale enterico NCC si crede di essere il principale fattore che contribuisce alla eziologia della malattia di Hirschsprung. Il colon, più lontana dalla fonte di migrazione NCC e ultima porzione di bowel ad essere colonizzato, è più suscettibile a difetti nella formazione ENS. In conformità con il suo ruolo cruciale nella migrazione enterico NCC, interruzione del segnale GDNF / RET è la principale causa conosciuta genetica della malattia di Hirschsprung 13. Per studiare meglio NCC e sviluppo ENS, abbiamo generato una linea di topi transgenici - chiamato Gata4p [5kb]-GFP 14 - in cui migratori NCC sono etichettati con la Green Fluorescent Protein (GFP). Abbiamo poi messo a punto un saggio ex vivo migrazione cellulare, adattato dal lavoro pubblicato da altri gruppi 11,12,15, che ora consente una precisa quantificazione della enterico potenziale migrazione NCC in presenza di vari fattori di crescita, quali GDNF.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

Cellule della cresta neurale (NCC) sono una popolazione transitoria e multipotenti cellule che proviene dal tubo neurale dorsale e migra ampiamente in tutto il vertebrato sviluppo dell&#39;embrione. Oltre a fornire glia e neuroni periferici, NCC generare melanociti nonché la maggior parte dello scheletro cranio-facciale. Migrazione NCC e la differenziazione è controllata da una combinazione della loro origine assiale lungo il tubo neurale e la loro esposizione ai segnali extracellulari regionale distinti. Tale contributo di ligandi extracellulari è particolarmente evidente durante la formazione del sistema nervoso enterico (ENS), una complessa rete interconnessa di gangli neurale che controlla localmente (tra le altre cose) movimento intestinale muscolare e la motilità intestinale. La maggior parte della ENS è derivato da una piccola piscina iniziale di NCC che intraprendere un lungo viaggio per colonizzare - in un rostrale alla moda caudale - l&#39;intera lunghezza del futuro dell&#39;intestino. Tra le varie vie di segnalazione noti perinfluenza enterico colonizzazione NCC, GDNF segnalazione / RET è riconosciuto come il più importante. Infatti, la secrezione controllata spatiotemporally della RET ligando GDNF dal mesenchima intestino è principalmente responsabile per l&#39;attrazione e la guida di-RET esprimere enterico NCC da e all&#39;interno dell&#39;intestino embrionale. Qui, descriviamo un ex vivo saggio migrazione cellulare, facendo uso di una linea di topi transgenici in possesso fluorescente NCC, che permette una precisa quantificazione della enterico potenziale migrazione NCC in presenza di vari fattori di crescita, tra GDNF.

I seguenti risultati sono rappresentativi di quello ottenibile con la tecnica descritta qui (Figura 1). L&#39;impiego di fattori di crescita (cioè GDNF) stimola la migrazione di GFP che esprimono enterico NCC dal espianto intestinale e nel gel di collagene (Figura 2). Sebbene alcune cellule escono espianto in assenza di fattori di crescita, questi non sono per lo GFP-etichettata e rappresentano entrata passiva. È necessario rimuovere la fetta intestinale dal gel di collagene ...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Presentiamo un ex vivo delle cellule saggio di migrazione che permette una precisa quantificazione della enterico potenziale migrazione delle cellule della cresta neurale in presenza di vari fattori di crescita.

Un saggio di migrazione cellulare quantitativa per murini enterici neurali Progenitori

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

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Research

JoVE Journal

Neuroscience

9.2K Views.  UQAM.  Presentiamo un ex vivo delle cellule saggio di migrazione che permette una precisa quantificazione della enterico potenziale migrazione delle cellule della cresta neurale in presenza di vari fattori di crescita. 

Video: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Un sottile cranio Tecnica Finestra per cronica a due fotoni In vivo</emImaging> della microglia nei modelli murini di neuroinfiammazione

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Ricerca

Neuroscienze

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Stabilire embrionali di topo Neural Stem Cell Culture del saggio di neurosfere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-formanti colonia saggio Cell: un test per discriminare Bona Fide cellule staminali neurali da cellule progenitrici neurali

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Analisi della migrazione cresta neurale cellulare Trunk con un Modified Zigmond Assay Camera

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Riproducibile nervo sciatico Crush mouse e successiva valutazione da parte della Rigenerazione intera analisi Muscle Monte

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Analisi della migrazione cresta neurale e la differenziazione delle specie da Cross-trapianto

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Il saggio neuroblasti: un test per la generazione e arricchimento delle cellule progenitrici neuronali di differenziare i Progeny cellule staminali neurali in citometria a flusso

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Un test Galvanotaxis Analisi della Cinetica precursori neurali migrazione cellulare in un campo applicato esternamente diretto Corrente elettrica

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Sviluppo di un protocollo di raffinata per Trans-sclerale Subretinal trapianto delle cellule epiteliali del pigmento retinico umano negli occhi di topo