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

1. Impostazione di base prima di procedere alla dissezione: <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> Il mezzo è riscaldato in un bagno d&#39;acqua a 37 ° C. </li><li> Freddo HEPES-buffered medio minimo indispensabile (HEM), con elevata concentrazione di antibiotici (10%) è preparato per la dissezione e lo scopo di lavaggio. In alternativa, media NSC basale con l&#39;integrazione di antibiotici può essere usato anche per questo scopo. </li><li> 25-30 ml di HEM freddo contenenti antibiotici è erogato in sterili 50 ml provette per la raccolta degli embrioni. </li><li> Diversi 10 centimetri di plastica piastre di Petri necessario a contenere gli embrioni e il cervello durante la dissezione e anche per tenere il tessuto sezionato. </li><li> Gli strumenti chirurgici, necessari per eliminare gli embrioni (forbici grandi, piccole forbici a punta, pinze grandi, piccole pinze curve) o per la dissezione del cervello embrionale (pinze piccolo, curvo pinza sottile, 45 ° angolo una pinza sottile, e forbicine) vengono sterilizzati con perle di vetro sterilizzatore a 250 ° C o altri metodi di autoclave disponibili. </li><li> Microscopio a dissezione è pulito con alcool al 70% e impostare all&#39;interno di una cappa a flusso laminare o PC2. </li></ol> 2. La raccolta E14 cervello di topo e Micro-dissezione: <ol><li> Una volta accoppiato il mouse incinta è anestetizzato al giorno 14 ° di gestazione secondo il proprio protocollo istituzionale animali approvati. </li><li> Dislocazione cervicale viene eseguita per assicurarsi che l&#39;animale non soffre il dolore e angoscia. </li><li> Il mouse anestetizzato è posto sulla sua schiena su una carta velina assorbente, e l&#39;addome è sciacquato con etanolo al 70% per sterilizzare la zona. </li><li> La pelle sopra l&#39;addome viene afferrata con una pinza di grandi dimensioni, e poi la pelle e la fascia sottostante è tagliato con le forbici di grandi dimensioni per esporre la cavità addominale e le corna uterine. </li><li> Cervice vengono rimossi con una pinza e forbici piccole e vengono trasferiti in una da 50 ml tubo contenente HEM freddo. </li><li> I tessuti uterino vengono trasferiti al cofano e poi sciacquate con una o due volte il volume abbastanza fresca HEM sterile fredda per rimuovere possibili contaminanti come il sangue e capelli. </li><li> I tessuti uterino vengono poi trasferiti su un piatto da 10 centimetri di Petri contenente HEM freddo. </li><li> Le corna uterine vengono aperti utilizzando piccole pinze e forbici curve e gli embrioni vengono trasferiti in un piatto di 10 centimetri nuova, che contiene HEM freddo. </li><li> Le teste degli embrioni sono separati a livello cervicale del midollo spinale e trasferito in un altro piastra Petri contenente HEM freddo. </li><li> La piastra Petri viene trasferita sotto un microscopio da dissezione per rimuovere il cervello dal cranio. </li><li> Le teste sono tenute con una pinza sottile curva dal lato caudale così come il lato dorsale delle teste sono rivolte verso l&#39;alto. Utilizzando micro-forbici, prima un taglio orizzontale è fatto sopra gli occhi per poi proseguire sulla linea mediana dalla fronte verso la parte posteriore della testa. Assicurati di tagliare attraverso la pelle e il cranio e non danneggiare il cervello sottostante. </li><li> Il cervello è rimosso dal cranio premendo i bordi della sezione di taglio con un movimento all&#39;indietro con le pinze curve. Questa procedura è continuata fino a quando tutti i cervelli vengono rimossi dal teschi. </li><li> Il cervello è tenuta ferma con le pinze curve in modo che il lato dorsale è rivolto verso l&#39;alto e quindi utilizzando microscissors viene eseguito un taglio attraverso la corteccia di ciascun emisfero si estende dal bulbo olfattivo alla parte posteriore dell&#39;emisfero per esporre le eminenze gangliari. </li><li> Utilizzando la pinza curva in una mano e il 45 ° angolo forcipe nell&#39;altra, i lembi taglio di cortecce sono sparsi e le eminenze gangliari vengono sezionati fuori. </li><li> Il tessuto sezionato viene inserito in una capsula di Petri sterile 10 centimetri, e questa procedura viene ripetuta fino a quando tutti i cervelli sono stati micro-sezionato. </li><li> Pezzi di tessuto sono raccolti con 1 ml di medium NSC in una pipetta da 1 ml di punta e trasferito in una provetta da centrifuga da 15 mL. </li><li> Tessuto è poi dissociato accuratamente, ma delicatamente, premendo la punta della pipetta sul fondo della provetta e pipettaggio la sospensione su e giù. In questo modo le macchie sono suddivisi in singole cellule. Pipettaggio viene eseguita 3-4 volte in modo che una sospensione lattiginosa come si ottiene. Poi, la sospensione è lasciata riposare per 1-2 minuti in modo da non dissociati grumi precipitati. </li><li> Quasi la sospensione cellulare viene trasferita l&#39;intera al tubo di altri e poi un altro 1 ml di medium NSC viene aggiunto al grumi rimasti e dissociato di singola cellula come descritto. </li><li> Il contenuto dei tubi sono raggruppati e centrifugato per 5 minuti a temperatura ambiente, a 700rpm (110g). </li><li> Il supernatante viene aspirata giù per l&#39;attuale pellet, e le cellule sono risospese in 1 ml di medium completo NSC. </li><li> Il mezzo è delicatamente pipettati su e giù peravere una sospensione omogenea singola cella. </li><li> 10 ml di sospensione cellulare viene miscelato con 90 ml di trypan blu per eseguire una conta cellulare. </li><li> Infine, le cellule sono placcati alla densità di 2x10 5 cellule / ml in piena medio NSC integrato con 20ng / mL di fattore di crescita epidermico (EGF). 5 ml di mezzo è usato per T25, 20 ml per il T75 e T175 40 ml per fiaschi. </li><li> Neurosfere si formano in 5-7 giorni se posti in incubazione a 37 ° C in un incubatore umidificato con 5% di CO 2. In 6-7 giorni, le sfere dovrebbe misurare tra 150-200 micron e sarà pronto per sottocultura (passaggio). </li></ol> 3. Passaging ed espansione di NSCs embrionali: <ol><li> Quando le neurosfere sono pronti per la subcultura (150-200 micron di diametro), il mezzo con sfere sospese viene rimossa dal fiaschi, collocati in un apposito tubo di tessuto sterile cultura dimensioni, e centrifugati a 700 giri (110 g) per 5 minuti a a temperatura ambiente. </li><li> Il supernatante viene scartato e le sfere sono risospesi in 1 ml di 0,05% tripsina-EDTA. </li><li> La sospensione cellulare viene quindi incubato in un bagno d&#39;acqua a 37 ° C per 2-3 minuti, poi un uguale volume di inibitore della tripsina di soia viene utilizzata per interrompere l&#39;attività tripsina. </li><li> La sospensione cellulare viene delicatamente pipettato su e giù per assicurare che la tripsina è stato completamente inattivato. </li><li> La sospensione cellulare viene centrifugata a 700 rpm (110g) per 5 min. Poi, il sopranatante viene rimosso e le cellule sono risospese in 1 ml di medium completo NSC. </li><li> 10 ml di sospensione cellulare viene miscelato con 90 ml di trypan blu per eseguire una conta cellulare. </li><li> Le cellule sono placcati in una concentrazione di 5x10 4 cellule / ml in totale medio NSC integrato con 20ng / mL di EGF in un apposito pallone di colture di tessuti dimensioni. 5 ml di mezzo è usato per T25, 20 ml per il T75 e T175 40 ml per fiaschi. </li><li> Neurosfere secondarie si formano in 5-7 giorni se posti in incubazione a 37 ° C in un incubatore umidificato con 5% di CO 2. </li></ol> 4. Rappresentante dei risultati: Nella scuola primaria la cultura embrionale NSC, la maggior parte delle cellule diventa ipertrofica e allegare ai piatti della cultura del tessuto su placcatura. Mentre la maggior parte delle cellule muoiono o differenziare, dopo 2-3 giorni, le cellule proliferative fare piccoli gruppi di cellule che si staccano dal substrato (Figura 1). Formazione di grandi aggregati sferoidali nelle prime 48 ore di cultura non deve essere confuso con sfere primarie. Formazione di aggregati dipende principalmente dalla quantità di detriti e non dissociata grumi di tessuto nella cultura. Neurosfere vero fase sono luminose e diventano più sferiche l&#39;aumento delle dimensioni (Figura 2). Microspikes piccolo appare sulla superficie esterna di sfere vitali e sani (Figura 3). Dopo 5-7 giorni, le sfere devono essere rotonda, ma non compattato, e dovrebbe misurare tra 150 e 200 micron di diametro. Se neurosfere sono autorizzati a crescere troppo grandi (dopo 9-10 giorni in coltura), potrebbero formare aggregati o diventare di colore scuro a causa della morte delle cellule al centro delle sfere (vedi video). Neurosfere di grandi dimensioni potrebbe finalmente cominciare a differenziare in situ (allegando al substrato e la migrazione verso la periferia). E &#39;anche difficile dissociare neurosfere di grandi dimensioni e sottocultura loro. <img src="/files/ftp_upload/2457/2457fig1.jpg" alt="Figura 1" /> Figura 1. Primaria E14 cultura NSC 3 giorni dopo placcatura. Frecce indicano i cluster proliferazione di NSC. Originale ingrandimento; 20x <img src="/files/ftp_upload/2457/2457fig2.jpg" alt="Figura 2" /> Figura 2. Primaria E14 cultura NSC 7 giorni dalla placcatura. Originale ingrandimento; 10x <img src="/files/ftp_upload/2457/2457fig3.jpg" alt="Figura 3" /> Figura 3. Passage uno E14 neurosfere 5 giorni dopo la placcatura. Si noti la micro-spikes (frecce) alla periferia delle sfere. Ingrandimento originale; 20x

<ol>
	<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>Morshead, C. M. <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+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</li><li>Craig, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+growth+factor+expansion+of+endogenous+subependymal+neural+precursor+cell+populations+in+the+adult+mouse+brain.">In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.</a> J Neurosci. 16, 2649-2658 (1996).</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>Azari, H., Rahman, M., Sharififar, S., BA, R. e. y. n. o. l. d. s. <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>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. 26, 988-996 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Il dosaggio neurosfere è il metodo di scelta per l&#39;isolamento e l&#39;espansione delle cellule staminali neurali 1-5 per la sua semplicità e riproducibilità. Questo saggio è uno strumento prezioso per la grande generazione di cellule precursori indifferenziati sistema nervoso centrale, che potrebbe essere utilizzato sia per in vitro e in vivo. Va sottolineato che neurosfere potrebbe essere generato sia dalla bona fide cellule staminali e progenitori neurali più ristretto. Pertanto, ...

* Per rendere HEM, mescolare 110L pacchetto di MEM and160ml di HEPES 1M e portare il volume a 8,75 L con acqua distillata. Impostare il PH finale a 7,4 e conservarlo a 4 ° C.

establishing embryonic mouse neural stem cell culture using the neurosphere assay

In mammiferi, le cellule staminali agisce come una fonte di cellule indifferenziate per mantenere la genesi e il rinnovamento cellulare in vari tessuti ed organi durante la vita dell&#39;animale. Essi possono potenzialmente sostituire le cellule che si perdono nel processo di invecchiamento o in via di infortuni e malattie. L&#39;esistenza di cellule staminali neurali (NSC) è stato descritto da Reynolds e Weiss (1992) negli adulti dei mammiferi sistema nervoso centrale (SNC) utilizzando un siero privo di sistema di coltura romanzo, il saggio neurosfere (NSA). Da questo test, è anche possibile isolare ed espandere NSCs provenienti da diverse regioni del SNC embrionale. Queste NSCs espanse in vitro sono multipotenti e possono dar luogo a tre tipi principali di cellule del sistema nervoso centrale. Mentre la NSA sembra relativamente semplice da eseguire, l&#39;attenzione alle procedure dimostrato qui è necessario per ottenere risultati affidabili e coerenti. Questo video dimostra praticamente NSA per generare ed espandere NSCs fin dal primo giorno embrionale 14-topo tessuto cerebrale e fornisce dettagli tecnici così si può raggiungere culture neurosfere riproducibile. La procedura prevede la raccolta E14 embrioni di topo, microdissezione cervello per raccogliere le eminenze gangliari, dissociazione dei tessuti raccolti in NSC medio per ottenere una sospensione singola cellula e, infine, placcatura cellule in coltura NSA. Dopo 5-7 giorni di cultura, la risultante neurosfere primarie sono diversi passaggi di espandere ulteriormente il numero delle NSCs per gli esperimenti futuri.

Watch this Scientific Journal Video about Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay at JoVE.com

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Questo protocollo video mostra l&#39;applicazione del test neurosfere per l&#39;isolamento e l&#39;espansione delle cellule staminali neurali da eminenze gangliari del giorno embrionale 14-topo cervello.

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 ...

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Research

JoVE Journal

Neuroscience

24.5K Views.  Shiraz University of Medical Sciences, Shiraz, Iran. Questo protocollo video mostra l'applicazione del test neurosfere per l'isolamento e l'espansione delle cellule staminali neurali da eminenze gangliari del giorno embrionale 14-topo cervello.

Video: Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Neuroscienze

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 ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

Proteine ​​localizzazione in 3D Cultura cellule staminali neurali: una metodologia di visualizzazione ibrida

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

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). ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Il trapianto chirurgico di cellule staminali neurali del mouse nel midollo spinale di topi infettati con virus dell&#39;epatite neurotropi mouse

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

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 ...

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. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolamento e la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

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