Diese Arbeit wurde durch Mittel aus dem Overstreet Foundation unterstützt.

1. Basic Set Up Bevor wir Dissection: <ol><li> Entsprechende Volumen der kompletten NSC Medium wird hergestellt, indem NeuroCult NSC Basal Medium und NeuroCult NSC Proliferation Supplements bei einem 9:1-Verhältnis, bzw. vorbereitet. </li><li> Das Medium wird in einem 37 ° C Wasserbad erwärmt. </li><li> Kalte HEPES-gepufferte Minimum Essential Medium (HEM) mit einer hohen Konzentration von Antibiotika (10%) ist für die Präparation und Waschen Zweck vorbereitet. Alternativ können NSC Basalmedium mit Antibiotika-Supplementierung ebenfalls für diesen Zweck verwendet werden. </li><li> 25-30 ml kaltem HEM mit Antibiotika ist in sterile 50 ml Röhrchen für Entnahme der Embryonen verzichtet. </li><li> Mehrere 10cm Petrischalen aus Kunststoff sind notwendig, um die Embryonen und Hirn bei der Präparation zu halten und auch seziert Gewebe zu halten. </li><li> Die chirurgische Instrumente, notwendig, um die Embryonen (große Schere, kleine spitze Schere, Pinzette große, kleine gebogene Pinzette) oder für die embryonale Gehirn Dissektion (kleine Pinzette, gebogen feinen Pinzette, 45 ° abgewinkelt feinen Pinzette und eine kleine Schere) zu entfernen sind sterilisiert mit Glasperlen Sterilisator bei 250 ° C oder anderen verfügbaren Autoklav-Verfahren. </li><li> Dissection Mikroskop ist mit 70% igem Alkohol abgewischt und im Inneren einer laminaren Strömung oder PC2 Haube gesetzt. </li></ol> 2. Harvesting E14 Maus Brain and Micro-Dissektion: <ol><li> Ein Mal gedeckt schwangere Maus ist am Tag betäubt den 14. Schwangerschaftswoche nach einem institutionellen genehmigt Tier-Protokoll. </li><li> Genickbruch wird durchgeführt, um sicherzustellen, dass das Tier nicht leidet Schmerzen und Leiden. </li><li> Die narkotisierten Maus auf dem Rücken auf einem saugfähigen Papiertuch gelegt, und der Bauch ist mit 70% Ethanol gespült, um den Bereich zu sterilisieren. </li><li> Die Haut über dem Bauch gegriffen mit einer großen Zange, und dann die Haut und das darunterliegende Faszie wird mit großer Schere geschnitten, um die Bauchhöhle und die Uterushörner aussetzen. </li><li> Die Uteri werden mit kleinen Zangen und Scheren entfernt und sind in einem 50-ml konischen Rohr-haltigen Kälte HEM übertragen. </li><li> Die uterine Gewebe sind an der Haube übertragen und dann ein-oder zweimal mit genügend Volumen von frischem sterilen kalten HEM gespült, um mögliche Verunreinigungen wie Blut und Haare zu entfernen. </li><li> Die uterine Gewebe werden dann zu einer 10cm Petrischale mit kaltem HEM übertragen. </li><li> Die Uterushörner werden geöffnet, indem kleine gebogene Pinzette und Schere und die Embryonen werden in eine neue 10cm Schüssel, die kalte HEM enthält übertragen. </li><li> Die Köpfe der Embryonen werden bei der zervikalen Rückenmarks Ebene aufgetrennt und auf eine andere Petrischale mit kaltem HEM. </li><li> Die Petrischale wird unter einem Binokular übertragen auf das Gehirn aus dem Schädel zu entfernen. </li><li> Die Köpfe sind mit einer feinen gebogenen Pinzette aus der kaudalen Seite, so der Rückenseite des Kopfes nach oben zeigen statt. Mit Mikro-Schere wird zunächst ein horizontaler Schnitt oberhalb der Augen gemacht und dann weiter in der Mittellinie von der Stirn auf die Rückseite des Kopfes. Vergewissern Sie sich durch die Haut und den Schädel geschnitten und nicht auf die zugrunde liegende Hirnschädigung. </li><li> Das Gehirn ist vom Schädel, indem Sie die Ränder der Schnittfläche in einer Rückwärtsbewegung mit den gebogenen Pinzette entfernt. Dieses Verfahren wird fortgesetzt, bis alle Gehirne aus dem Schädel entfernt werden. </li><li> Das Gehirn ist stabil gehalten mit dem gebogenen Pinzette, so dass die Rückenseite nach oben und dann mit Blick über Mikroschere einen Schnitt durch die Rinde der beiden Hemisphären, die sich von den Riechkolben an der Rückseite der Hemisphäre zu den Ganglien-Eminenzen aussetzen durchgeführt wird. </li><li> Mit der gebogenen Pinzette in der Hand und der 45 ° abgewinkelte Pinzette in der anderen werden die geschnittenen Klappen Kortex verteilt und den Ganglien-Eminenzen sind herauspräpariert. </li><li> Die sezierten Gewebes wird in einer sterilen 10cm Petrischale gelegt, und dieser Vorgang wird solange wiederholt, bis alle die Gehirne wurden Mikro-seziert. </li><li> Gewebestücke werden gesammelt, indem 1 mL der NSC-Medium in einem 1 mL Pipette und in ein 15 mL Zentrifugenröhrchen. </li><li> Das Gewebe wird dann dissoziiert gründlich, aber sanft, indem Sie die Pipettenspitze auf den Boden der Röhre und Pipettieren der Suspension auf und ab. Auf diese Weise die Klumpen sind in einzelne Zellen gebrochen. Pipettieren ist 3-4 mal so, dass eine milchige wie Federung erreicht wird durchgeführt. Dann wird die Suspension für 1-2 Minuten so zufrieden wie die nicht-dissoziierten Klumpen ausfällt. </li><li> Fast die gesamte Zellsuspension wird auf die andere überführt und dann noch 1 ml NSC Medium ist, um die verbleibenden Klumpen und dissoziiert einzelne Zelle wie oben beschrieben aufgenommen. </li><li> Der Inhalt der Rohre werden gepoolt und für 5 Minuten bei Raumtemperatur zentrifugiert, bei 700rpm (110g). </li><li> Der Überstand wird abgesaugt bis zur eigentlichen Pellet, und die Zellen werden in 1 ml komplettem NSC-Medium resuspendiert. </li><li> Das Medium wird sanft auf und pipettiert biseine homogene einzigen Zellsuspension. </li><li> 10 ul der Zellsuspension mit 90 ul von Trypanblau auf eine Zellzahl führen gemischt. </li><li> Schließlich werden die Zellen bei der Dichte von 2x10 5 Zellen / ml in kompletten NSC Medium mit 20ng / ml epidermalen Wachstumsfaktor (EGF) ausplattiert. 5 ml Medium für T25, 20 mL für T75 und 40 mL für T175 Flaschen verwendet. </li><li> Neurosphären sind in 5-7 Tagen gebildet werden, wenn bei 37 ° C in einem befeuchteten Inkubator mit 5% CO 2 inkubiert. In 6-7 Tagen sollte die Sphären zwischen 150-200 Mikrometer messen und bereit sein wird, Subkultur (Durchgang). </li></ol> 3. Die Passage und Expansion von embryonalen NSCs: <ol><li> Wenn die Neurosphären bereit für Subkultur (150-200 &amp;mgr; m Durchmesser) sind, ist das Medium, mit hängenden Kugeln aus den Flaschen entfernt, in einer angemessenen Größe sterile Zellkulturgefäß und zentrifugiert bei 700 rpm (110 g) für 5 min bei Raumtemperatur. </li><li> Der Überstand wird verworfen und die Kugeln sind in 1 ml% 0,05 Trypsin-EDTA resuspendiert. </li><li> Die Zellsuspension wird dann in einem 37 ° C Wasserbad für 2-3 min inkubiert, dann ein gleiches Volumen an Sojabohnen-Trypsin-Inhibitor verwendet wird, um das Trypsin zu stoppen. </li><li> Die Zellsuspension wird vorsichtig nach oben und unten pipettiert, um sicherzustellen, dass die Trypsin wurde komplett inaktiviert. </li><li> Die Zellsuspension wird bei 700 rpm (110g) für 5 min zentrifugiert. Dann wird der Überstand abgenommen und die Zellen werden in 1 ml komplettem NSC-Medium resuspendiert. </li><li> 10 ul der Zellsuspension mit 90 ul von Trypanblau auf eine Zellzahl führen gemischt. </li><li> Die Zellen werden in einer Konzentration von 5x10 4 Zellen / ml in kompletten NSC Medium mit 20ng / ml EGF in einer angemessenen Größe Zellkulturflasche ausplattiert. 5 ml Medium für T25, 20 mL für T75 und 40 mL für T175 Flaschen verwendet. </li><li> Sekundäre Neurosphären sind in 5-7 Tagen gebildet werden, wenn bei 37 ° C in einem befeuchteten Inkubator mit 5% CO 2 inkubiert. </li></ol> 4. Repräsentative Ergebnisse: In primären embryonalen NSC Kultur, wird die Mehrheit der Zellen hypertrophe geworden und heften sich an das Gewebe Kulturschalen auf Plattierung. Während die Mehrheit der Zellen entweder sterben oder zu differenzieren, nach 2-3 Tagen, um proliferative Zellen kleine Gruppen von Zellen, die aus dem Substrat (Abbildung 1) lösen wird. Bildung von großen kugelförmigen Aggregaten in den ersten 48-Stunden-Kultur sollte nicht für die primäre Sphären verwechselt werden. Aggregatbildung hängt hauptsächlich von der Mengen von Schutt und nicht-dissoziierten Gewebe Klumpen in der Kultur. Wahre Neurosphären werden Phase hell und werden mehr als sphärische Größe zunimmt (Abbildung 2). Kleine microspikes erscheint auf der äußeren Oberfläche des lebensfähig und gesund Kugeln (Abbildung 3). Nach 5-7 Tagen, müssen die Kugeln rund sein, aber nicht verdichtet, und sollte zwischen 150 und 200 um im Durchmesser messen. Wenn Neurosphären erlaubt sind zu groß werden (nach 9-10 Tagen in Kultur), könnten sie Aggregate bilden oder dunkel in der Farbe, weil der Zelltod in der Mitte der Kugeln (siehe Video). Große Neurosphären könnten irgendwann anfangen zu in situ zu unterscheiden (Befestigung auf dem Substrat und die Migration in Richtung der Peripherie). Es ist auch schwierig, große Neurosphären distanzieren und Subkultur sie. <img src="/files/ftp_upload/2457/2457fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Primary E14 NSC Kultur 3 Tage nach dem Ausplattieren. Die Pfeile zeigen die proliferierenden Cluster von NSCs. Original-Vergrößerung; 20x <img src="/files/ftp_upload/2457/2457fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Primäre E14 NSC Kultur 7 Tage nach dem Ausplattieren. Original-Vergrößerung, 10x <img src="/files/ftp_upload/2457/2457fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Gang sind E14 Neurosphären 5 Tage nach dem Ausplattieren. Beachten Sie die Mikro-Spikes (Pfeile) an der Peripherie der Kugeln. Original-Vergrößerung, 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>

Die Neurosphäre Test ist die Methode der Wahl für die Isolierung und Expansion von neuralen Stammzellen 1-5 wegen seiner Einfachheit und Reproduzierbarkeit. Dieser Assay ist ein unschätzbares Werkzeug für Großanlagen von undifferenzierten Vorläuferzellen ZNS, die sowohl für in vitro und in vivo-Studien verwendet werden könnte. Es sollte betont werden, dass Neurosphären sowohl von der bona fide neuralen Stammzellen und mehr eingeschränkt Vorfahren konnten generiert werden kann. Dahe...

* Um HEM machen, mischen 110L Paket MEM and160ml 1M HEPES und bringen das Volumen auf 8,75 L mit destilliertem Wasser. Setzen Sie den End-pH auf 7,4 und speichern Sie sie bei 4 ° C.

establishing embryonic mouse neural stem cell culture using the neurosphere assay

In Säugetieren wirkt Stammzellen als Quelle von undifferenzierten Zellen zu Zelle Entstehung und Erneuerung in verschiedenen Geweben und Organen während der Lebensdauer des Tieres zu erhalten. Sie können möglicherweise ersetzen Zellen, die in den Alterungsprozess oder in den Prozess der Verletzungen und Krankheiten verloren gehen. Die Existenz von neuralen Stammzellen (NSCs) wurde zuerst von Reynolds und Weiss (1992) in der erwachsenen Säugetieren zentralen Nervensystems (ZNS) mit einem neuartigen serumfreien Kultur-System, das Neurosphäre Assay (NSA) beschrieben. Unter Verwendung dieses Tests ist es auch möglich zu isolieren und zu erweitern NSCs aus verschiedenen Regionen des embryonalen ZNS. Diese in vitro erweitert NSCs sind multipotente und kann zu den drei wichtigsten Zelltypen des ZNS geben. Während die NSA scheint relativ einfach durchzuführen, um die Verfahren demonstriert hier Aufmerksamkeit erforderlich ist, um verlässliche und konstante Ergebnisse zu erzielen. Dieses Video zeigt praktisch NSA zu erzeugen und zu erweitern NSCs aus embryonalen Tag 14-Maus-Gehirngewebe und erbringt technische Details so dass man reproduzierbare Neurosphäre Kulturen zu erreichen. Das Verfahren schließt die Gewinnung E14 Mausembryonen, Gehirn Mikrodissektion zu den Ganglien-Eminenzen, Dissoziation des geernteten Gewebes in NSC Medium Ernte einer einzigen Zellsuspension zu gewinnen, und schließlich plating Zellen in NSA Kultur. Nach 5-7 Tagen in Kultur, sind die resultierenden primären Neurosphären passagiert weiter ausbauen die Anzahl der NSCs für zukünftige Experimente.

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

Dieses Video-Protokoll zeigt die Anwendung der Neurosphäre Assay für die Isolierung und Expansion von neuronalen Stammzellen, die aus den Ganglien-Eminenzen der embryonalen Tag 14-Maus-Gehirn.

Gründung Embryonale Maus Neural Stem Cell Culture Mit dem Neurosphäre 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 ...

establishing-embryonic-mouse-neural-stem-cell-culture-using

Research

JoVE Journal

Neuroscience

24.5K Aufrufe.  Shiraz University of Medical Sciences, Shiraz, Iran. Dieses Video-Protokoll zeigt die Anwendung der Neurosphäre Assay für die Isolierung und Expansion von neuronalen Stammzellen, die aus den Ganglien-Eminenzen der embryonalen Tag 14-Maus-Gehirn.

Serie: 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.

Dissection und Kultur kommissuralen Nervenzellen aus embryonalen Rückenmark

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

Forschung

Neurowissenschaften

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.

Isolation und Expansion der erwachsenen Maus Neural Stem Cells mit dem Neurosphäre 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 ...

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.

Lokalisieren von Protein in 3D Neural Stem Cell Culture: Ein Hybrid Visualisierung Methodology

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-Colony Forming Zell-Assay: Ein Test zur Bona Fide neuralen Stammzellen aus neuralen Vorläuferzellen Discriminate

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.

Chirurgische Transplantation von Maus Neural Stem Cells in den Rückenmark von Mäusen mit neurotropen Maus Hepatitis Virus infiziert

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.

Die Neuroblast Assay: Ein Test für die Erzeugung und Anreicherung von neuronalen Vorläuferzellen aus neuralen Stammzellen differenzieren Progeny mittels Durchflusszytometrie

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.

Isolierung und Kultivierung von Zellen der Neuralleiste aus embryonalen murinen Neuralrohr

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.

Enumeration neuraler Stammzellen verwenden Geklonte 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.

Isolierung und Kultur von adulten neuralen Stammzellen aus der Maus subcallosa Zone

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.

Entwicklung eines raffinierten Protokolls für Trans-skleralen subretinalen Transplantation von menschlichen retinalen Pigment Epithelzellen in Ratte Augen