Questo lavoro è stato sostenuto da un finanziamento della Fondazione Overstreet.

1. Elementi che devono essere preparate prima di procedere alla placcatura Cell: <ol><li> Volume appropriato di completo media NSC è preparata mescolando NeuroCult medio basale NSC e NeuroCult NSC Supplementi proliferazione con un rapporto 9:1, rispettivamente. </li><li> Un&#39;aliquota di NeuroCult NCFC mezzo privo di siero senza citochine è scongelato. </li><li> Il mezzo è riscaldato in un bagno d&#39;acqua a 37 ° C. </li><li> Soluzioni stock di fattore di crescita epidermico (EGF), fattore di crescita fibroblastico base (b-FGF) alla concentrazione di 10 mg / ml ed eparina a 0,2% sono preparati in anticipo. </li><li> A seconda delle dimensioni esperimento, 35 millimetri diversi piatti colture di tessuti sono necessari per la piastra di cellule e un piatto 150-200cm plastica Petri è necessario anche per tenere i piatti duplicare 35mm e un piatto terza 35mm per l&#39;acqua. </li></ol> 2. Preparazione delle cellule: A seconda del vostro esperimento, le cellule possono essere preparati da una fonte adulte o embrionali (primaria del tessuto dissociati o neurosfere dissociato). Sezionare tessuti di adulti / embrionale del mouse sistema nervoso centrale (SNC) o dissociarsi neurosfere adulto / embrionali derivate, come descritto prima 1,2 e poi: <ol><li> La sospensione singola cellula viene fatta passare attraverso un 40-micron di dimensioni filtro a rete in modo da eliminare non dissociati grumi. </li><li> 10μl di sospensione cellulare viene miscelato con 90μl di Blu tripano per eseguire una conta cellulare. Nota: altre diluizioni cella appropriata può anche essere usato. </li><li> Se si utilizza embrionale primario o adulto cellule derivate neurali, diluire la sospensione cellulare ad una concentrazione di 6,5 x 10 5 cellule / ml in totale medio NSC. Se si utilizzano le cellule dissociate da neurosfere derivate da cellule neurali adulte o embrionali, diluire la sospensione cellulare ad una concentrazione di 2,2 x 10 5 cellule / ml in totale medio NSC. </li></ol> 3. Cellule placcatura in semi-solido N-ACCP Media: <ol><li> Il volume appropriato di seguenti componenti è mescolato in ordine, a seconda del numero di repliche. Qui mescolare la quantità necessaria per due repliche o duplicare i piatti. Per gli altri numeri delle repliche, si prega di fare riferimento alla tabella 1. <ul><li> 1,7 ml di NeuroCult NCFC mezzo privo di siero senza Citochine </li><li> 330 ml di NeuroCult NSC Supplementi proliferazione </li><li> 6,6 ml di EGF (10μg/mL) </li><li> 32 ml di penicillina / streptomicina </li><li> 3,3 ml di b-FGF (10μg/mL) necessaria solo se le cellule derivate da colture di cellule neurali adulte. </li><li> 3,3 ml di eparina (0,2%) hanno richiesto solo se le cellule derivate da colture di cellule neurali adulte. </li><li> 25μL di sospensione cellulare (di 2,2 x 10 5 cellule / ml cellule da neurosfere dissociati o 6,5 x 10 5 cellule / ml cellule primarie da dissociato tessuto del SNC). Nota: I numeri di cellulare placcato finale dovrebbe essere circa 2500 cellule per 35 millimetri piatto cultura per neurosfere cellule derivate e 7500 cellule per 35 mm per piatto cultura cellule primarie. In alcuni casi, la densità finale delle cellule placcatura deve essere regolato eseguendo un esperimento di titolazione delle cellule. Per le analisi statistiche, il numero totale di colonie rilevate dopo 21 giorni di cultura dovrebbe essere entro l&#39;intervallo di almeno 50-150 colonie. </li></ul></li><li> Il mezzo che contiene le cellule si mescola delicatamente e poi 1,3 mL di soluzione di collagene freddo è trasferito alla sospensione cellulare e mescolato bene pipettando delicato per evitare l&#39;introduzione di eventuali bolle. </li><li> La soluzione mista è dispensato al centro di ogni piatto cultura 35mm (~ 1,5 ml / piatto) ei piatti sono punta delicatamente con un movimento circolare per lasciare che la miscela diffuse uniformemente sulla superficie del piatto. . </li><li> Il duplicato 35 piatti cultura mm sono poste in un piatto di 100 mm Petri. Il coperchio del piatto nuovo 35 mm è rimosso e il piatto è aperto anche messo nel piatto stessi 100 millimetri Petri. Acqua sterile viene aggiunto a questo open piatto 35 mm a mantenere l&#39;umidità ottimale durante il periodo di incubazione. Piastre di test biologico piazza (245 mm) vengono utilizzati quando più replicare piatti 35 mm sono stati istituiti. Ancora una volta, comprendono 2 o 3 piatti aperta 35 mm contenente acqua sterile. </li><li> Le piastre sono trasferiti in un incubatore a 37 ° C, 5% di CO2 e il 95% di umidità. Il rapprende collagene da aumento della temperatura e la formazione di gel avverrà entro circa un&#39;ora. Le culture non deve essere disturbato durante questo periodo. </li><li> Cellule coltivate vengono incubate per 21 giorni (differenze di dimensioni della colonia può essere chiaramente distinta dopo 21 giorni). </li></ol> 4. Preparazione media di ricarica e di alimentazione della Cultura: Come N-ACCP culture vengono incubate per un lungo periodo di tempo (21 giorni), le colture devono essere alimentati con il NeuroCult appropriato completo medio di riempimento preparato fresco come segue: <ul><li> 4,5 ml di Medium NeuroCult NSC basale è mescolato con 0,5 mL di NeuroCult NSC Supplementi proliferazione e poi i fattori di crescita si aggiunge: </li><li> 250 microlitri di 10μg/mL EGF (per ottenere una concentrazione finale di 0,5 mg / ml FEG) </li><li> 125 ml di 10μg/mL b-FGF (per ottenere una concentrazione finale di 0.25μg/mL b-FGF) richiesto solo se le cellule derivate da colture di cellule neurali adulte. </li><li> 125 ml di eparina 0,2%, necessario solo se le cellule derivate da colture di cellule neurali adulte. </li><li> 60 ml di Medium appropriato completa Replenishment NeuroCult (per le cellule embrionali o adulte) viene aggiunto al centro di ogni piatto saggio NCFC una volta ogni 7 giorni durante l&#39;incubazione intero NCFC cultura Assay (21 giorni). </li></ul> 5. Punteggio N-CFC Colonie Assay derivati ​​e calcolo della frequenza di bona fide NSCs e cellule progenitrici neurali: <ul><li> Ogni piatto 35 mm è la cultura messo su un piatto punteggio griglia con la dimensione della griglia di 2,0 mm x 2,0 mm e poi entrambi i piatti sono immessi sul palco microscopio. </li><li> Utilizzando bassa potenza (2.5X - 5X) lente dell&#39;obiettivo, ogni piatto viene sottoposto a scansione e le colonie sono segnati in base alle loro dimensioni. </li><li> Le colonie sono classificati in due categorie principali: <ol><li> Meno di 2 mm di diametro </li><li> ≥ 2 mm di diametro </li></ol></li></ul> 6. Rappresentante dei risultati: Come in test neurosfere, le cellule placcato in N-CFC test iniziano a proliferare e fare piccole colonie di cellule entro 3 - 7 giorni dopo la placcatura (Figura 1). Tra due settimane, le colonie di diverse dimensioni si possono distinguere. Mentre la maggior parte delle colonie tendono a smettere di crescere dopo 14 giorni, alcune colonie continuano a crescere di dimensione. Dopo 21 giorni, le colonie possono essere classificati in una delle quattro categorie: 1) meno di 0,5 mm di diametro, 2) 0.5 - 1 mm di diametro, 3) 1 - 2 mm di diametro e 4) ≥ 2 mm di diametro. In pratica, le colonie di dimensioni inferiori a 2 mm di diametro sono indicati come progenitrici derivate (Figura 2) e colonie ≥ 2 millimetri di diametro sono indicati come NSC derivati ​​(Figura 3). Il numero di colonie ≥ 2 millimetri di diametro per cellule totali placcato, rappresenta la frequenza del reale in buona fede le cellule staminali neurali a lungo termine di auto-rinnovamento e multi-potenziali capacità. Le neurosfere totale formando frequenza in una popolazione di cellule particolare dopo 7-8 giorni in un esperimento parallelo NSA è stata stimata essere simile al totale delle colonie che formano la frequenza della popolazione stessa cella dopo 21 giorni in un N-ACCP esperimento. L&#39;N-ACCP tuttavia, costituisce una condizione più permissivo in modo che ogni cellula può mostrare tutto il suo potenziale proliferativo. <table border="1"><tbody><tr><td> Componente </td><td> 2 si replica </td><td> 3 repliche </td><td> 4 repliche </td></tr><tr><td> NeuroCult NCFC mezzo privo di siero senza Citochine </td><td> 1700 </td><td> 2550 </td><td> 3400 </td></tr><tr><td> NeuroCult NSC proliferazione Supplementi </td><td> 330 </td><td> 495 </td><td> 660 </td></tr><tr><td> EGF (10 mg / ml) </td><td> 6,6 </td><td> 9,9 </td><td> 13,2 </td></tr><tr><td> bFGF (10 mg / ml), solo per le cellule adulte </td><td> 3,3 </td><td> 4,95 </td><td> 6,6 </td></tr><tr><td> Soluzione di eparina (0,2%), solo per le cellule adulte </td><td> 3,3 </td><td> 4,95 </td><td> 6,6 </td></tr><tr><td> Penicillina / streptomicina (1:100) </td><td> 32 </td><td> 48 </td><td> 64 </td></tr><tr><td> Celle a: 2,2 x 10 5 colture di cellule / ml O 6,5 x 10 5 cellule primarie / mL </td><td> 25 </td><td> 37,5 </td><td> 50 </td></tr><tr><td> Collagene Soluzione </td><td> 1300 </td><td> 1950 </td><td> 2600 </td></tr></tbody></table> Tabella 1. Componenti di completo N-CFC cultura test. <img src="/files/ftp_upload/2639/2639fig1.jpg" alt="Figura 1" /> Figura 1. Colonie Rappresentante in N-ACCP cultura di passaggio uno E14 NSCs del mouse 7 giorni dalla placcatura. Le colonie potrebbe visualizzare la morfologia e dimensioni diverse. Originale ingrandimento; 4x <img src="/files/ftp_upload/2639/2639fig2.jpg" alt="Figura 2" /> Figura 2. Colonie progenitrici derivate rappresentante di diverse dimensioni in N-ACCP cultura di passaggio del mouse uno E14 NSCs 21 giorni dopo placcatura. Come si vede, le colonie hanno una morfologia diversa, ma tutti sono 2 mm in meno di dimensione. Originale ingrandimento; 4x <img src="/files/ftp_upload/2639/2639fig3.jpg" alt="Figura 3" /> Rappresentante Figura 3. Bona fide di cellule staminali derivate colonia in N-ACCP cultura di passaggio del mouse uno E14 NSCs 21 giorni dopo placcatura. Colonie di cellule staminali potrebbe visualizzare diversa morfologia (vediil video) ma tutti sono ≥ 2 millimetri di diametro. Originale ingrandimento; 4x

<ol>
	<li>Azari, H., Rahaman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J Vis Exp. , (2010).</li><li>Azari, H., Sharififar, S., Rahaman, M., Ansari, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J Vis Exp. , (2011).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+Neural+Stem+and+Progenitor+Cells+in+the+Neural+Colony+Forming+Cell+Assay.">Enumeration of Neural Stem and Progenitor Cells in the Neural Colony Forming Cell Assay.</a> Stem Cells. , (2008).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li><li>Louis, S. A., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+and+Neural+Colony-Forming+Cell+Assays.">Neurosphere and Neural Colony-Forming Cell Assays.</a> Protocols for Neural Cell Culture. 10, 1-28 (2010).</li></ol>

Uno degli autori, Louis A. Sharon, è affiliata con StemCell Technologies Inc, produttore di alcuni reagenti usati in questo studio.

Sebbene saggio neurosfere 3,1,2 è il metodo più comune per isolare ed espandere cellule staminali neurali da una varietà di fonti come adulto che embrionale del tessuto nervoso centrale, non può misurare con precisione la frequenza di NSC in una popolazione mista di cellule precursori neurali (staminali e progenitori), come non c&#39;è una relazione uno a uno tra il numero di neurosfere e il numero di cellule staminali in buona fede 4. Per far fronte a questa limitazione, l&#39;originale NSA è stato adattato in modo da consentire il staminali neurali e cellule progenitrici di crescere la loro capacità di proliferazione pieno per tre settimane 5-7. A differenza del liquido NSA, in N-ACCP le colonie sono in realtà clonale derivati, come il semi-solida matrice di collagene impedisce la migrazione delle cellule singole placcato e l&#39;aggregazione delle colonie. Per ottenere risultati coerenti con questa analisi si consiglia: <ol><li> Garantire una sospensione singola cella della sospensione cellulare originale. Far passare la sospensione singola cellula con un 40-micron di dimensioni filtro a rete in modo da eliminare non dissociati grumi. </li><li> Conservare la soluzione di collagene su ghiaccio o in frigorifero C ° +4. Il collagene è l&#39;ultimo elemento da aggiungere alla miscela di sospensione cellulare come si congela aumentando la temperatura. </li><li> Non dimenticare di alimentare la cultura ogni settimana. Aggiunta del supporto di rifornimento che contiene il fattore di crescita (s) una volta ogni 7 giorni permette alle cellule di continuare a proliferare nel lungo periodo la cultura. </li><li> La densità finale delle cellule placcatura sono stati ottimizzati per le cellule derivate da cellule neurali primarie o coltivate in modo che il numero totale di colonie rilevate dopo 21 giorni di cultura nel NCFC-A è nel range di almeno 50-200 colonie. Questa gamma permette di individuare le rare colonie NSC derivati&gt; 2mm di diametro, in campioni, fornendo un numero sufficiente di colonie per analisi statistiche. A seconda della sorgente di cellule di partenza, un numero significativamente inferiore (&lt;50) e un numero maggiore di colonie (&gt; 250) sono a volte ottenuti. Colonie troppo pochi comporterà l&#39;&gt; colonie 2mm di diametro al di sotto dei livelli di rilevamento, mentre le colonie troppi si tradurrà in anddepletion sovraffollamento dei componenti mezzo di crescita con un conseguente conteggio delle colonie inesatte. Pertanto la densità delle cellule placcatura dovranno essere adeguati di conseguenza (cioè aumentando o diminuendo il numero totale di cellule placcato) </li></ol>

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<td>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05700</td>
<td>&nbsp;</td>
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<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>STEMCELL</td>
<td>05701</td>
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<td>0.05% 	trypsin-EDTA</td>
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<td>Gibco</td>
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<td>Gridded scoring dishes</td>
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neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells

Il test neurosfere (NSA) è uno dei metodi più frequentemente utilizzati per isolare, espandere e anche calcolare la frequenza di cellule staminali neurali (NSC). Inoltre, questo siero privo di sistema di coltura è stato utilizzato per espandere le cellule staminali e determinare la loro frequenza da una varietà di tumori e tessuti normali. E &#39;stato dimostrato recentemente che uno-a-uno non esiste tra formazione neurosfere e NSC. Questo suggerisce che la NSA come attualmente applicato, sovrastima la frequenza di NSCs in una popolazione mista di cellule precursori neurali isolate dal cervello sia embrionali che adulte dei mammiferi. Questo video dimostra praticamente un romanzo a base di collagene semi-solido saggio, il neuro-formanti colonia test delle cellule (N-ACCP), che ha la capacità di discriminare staminali da cellule progenitrici in base al loro lungo termine potenziale proliferativo e, quindi, fornisce un metodo per enumerare frequenza NSC. Nella N-ACCP, colonie ≥ 2 mm di diametro sono derivati ​​da celle che soddisfano tutti i criteri funzionali di NSC, mentre le colonie &lt;2mm sono derivati ​​da progenitori. L&#39;N-ACCP procedura può essere utilizzata per le cellule preparate da fonti diverse tra cui adulti primarie e colta o embrionali sistema nervoso centrale del mouse cellule. Qui usiamo le cellule preparate dal passaggio uno neurosfere generato dal primo giorno 14 embrionale del cervello nei topi per eseguire N-ACCP. Le culture sono rifornito con la proliferazione di media ogni sette giorni per tre settimane per consentire alle cellule placcato ad esporre il loro pieno potenziale proliferativo e quindi la frequenza delle progenitrici neurali e in buona fede le cellule staminali neurali è calcolato rispettivamente contando il numero di colonie che sono &lt;2mm e quelli che sono ≥ 2 millimetri in riferimento al numero di cellule che sono stati inizialmente placcato.

Watch this Scientific Journal Video about Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells at JoVE.com

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

Questo protocollo video viene illustrato come discriminare ed enumerare in buona fede le cellule staminali neurali in una popolazione mista di cellule precursori neurali utilizzando il neurali che formano colonie test cell.

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). ...

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Neuroscience

22.0K Views.  University of Florida. Questo protocollo video viene illustrato come discriminare ed enumerare in buona fede le cellule staminali neurali in una popolazione mista di cellule precursori neurali utilizzando il neurali che formano colonie test cell.

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolamento ed espansione della cellule staminali neurali del mouse utilizzando il saggio neurosfere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

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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 ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

Un metodo di espansione cGMP-applicabile per Aggregati di neurali umane staminali e cellule progenitrici derivate da cellule staminali pluripotenti o fetale tessuto cerebrale

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivazione libero-Feeder della cresta neurale progenitrici Cellule pluripotenti da cellule staminali umane

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Ordinamento delle cellule staminali neurali e di cellule progenitrici del topo adulto subventricolare Zone e Live-imaging loro dinamiche del ciclo cellulare

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Conteggio di cellule staminali neurali L&#39;utilizzo clonali Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

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