Wir danken Evan Dysart und Marc Léonard für technische Unterstützung durch Experten in der Videoproduktion und-bearbeitung, Matt Cooke für die Unterstützung bei der Datenerhebung und Sarah Gelbard für Experten redaktionelle Unterstützung. Die Arbeit wurde durch Betriebskostenzuschüsse von der Canadian Institute of Health Research (CIHR, MOP 62626) zu SALB, eine strategische Training Initiative in Health Research Programm zu gewähren SALB und SF (CIHR Training Program in Neurodegenerative Lipidomics, TGF 9121), und die Infrastruktur finanziert Unterstützung von der kanadischen Stiftung Innovation und Ontario Innovation Trust SF. SI erhielt Ontario Graduate Scholarship in Wissenschaft und Technologie mit Beiträgen der Parkinson Research Consortium. NV erhält Institute of Aging und CIHR Training Program in Neurodegenerative Lipidomics post-professionellen Gemeinschaft. Wir danken für die Bildungs-Software und technischen Support durch Autodesk Forschung zur Verfügung gestellt.

<ol>
	<li>Kempermann, G., Jessberger, S., Steiner, B., Kronenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milestones+of+neuronal+development+in+the+adult+hippocampus.">Milestones of neuronal development in the adult hippocampus.</a> Trends Neurosci. 27, 447-452 (2004).</li><li>Campos, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurospheres:+insights+into+neural+stem+cell+biology.">Neurospheres: insights into neural stem cell biology.</a> J Neurosci Res. 78, 761-769 (2004).</li><li>Imbeault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+controls+gap+junction+protein+expression+and+function+in+postnatal+hippocampal+neural+progenitor+cells.">The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells.</a> BMC Neurosci. 10, 13-13 (2009).</li></ol>

Alle Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von der University of Ottawa Animal Care Committee und der Canadian Council on Animal Care Set durchgeführt.

Dieses Protokoll beschreibt ein Verfahren zur Kultur und seriell Gefrier postnatalen Hippocampus NPCs, lokalisieren Proteinexpression durch Immunodetektion und schließlich zu rekonstruieren und zu analysieren, die topographische Lage der immunpositive Zellen innerhalb des gesamten 3D-Neurosphäre. Durch die Kombination zellbiologischer Kultur-und Verarbeitungstechniken, Mikroskopie (OpenLab, Improvisation und andere Bildanalyse Software), Grafik-Editing-Software-Kapazitäten (Adobe Photoshop) und 3D-Animation und Compositing-Software-Kapazitäten (Autodesk Maya), präsentieren wir eine Methode zur Rekonstruktion gesamte zelluläre Zusammensetzung von 3D-Kulturen aus serielles 2D-Bilder ermöglicht die originalgetreue Rekonstruktion der zellulären Position und Protein-Expression während einer Neurosphäre Kultur. Dieses Protokoll kann mit der gleichen Effektivität, um die Registrierung und die Rekonstruktion der epifluoreszenten dünne Schnittserien oder konfokalen Z-Stapel durch dickere Schnittserien kombiniert werden. Da klonale Expansion einzelner NPCs in Neurosphäre Kultur rekapituliert viele der Stufen im Laufe der Neurogenese und Gliogenese in postnatalen Gehirn beobachtet, stellt die Auswirkungen seiner 3D-Struktur ein wichtiger NPC Schicksal bestimmend in Nachkommen ausgesetzt, um die gleichen experimentellen Milieu 1. Divergenz in Linie und Protein-Expression im Kern und Peripherie von seriellen Neurosphäre Abschnitte darauf hin, dass mehrere physische Faktoren wie Zell Lage, mechanische Belastung und Querkraft spielen eine wichtige Rolle bei der Regulierung der NPC Biologie 2. Darüber hinaus hat Connexin-Protein-Expression, hier analysierten, wurde gezeigt, dass sich je nachdem, wann NPCs sind als 3D-Neurosphären in Suspension oder vergoldet auf Laminin Substrat mit funktionellen Connexin-vermittelter Kommunikation verändert, ob die Zellen auf Kunststoff-und Substrat oder in 3D-frei sind kultivierte kultivierten -schwimmende Kulturen 3. Die Auswirkungen der zellulären Position auf NPC Schicksal bleibt unklar. Es ist technisch anspruchsvoll, um Auswirkungen der räumlichen Lage innerhalb 3D-Kultur zu analysieren auf NPC Biologie gegeben, dass antigenen Beurteilung der Abstammung und Signalproteine ​​Störung der 3D-Architektur zu Zellpermeabilisierung und Antikörper-Zugriff auf alle Zellen zu aktivieren muss. Hier zeigen wir, dass ein Hybrid-Visualisierung Methodik ein Mittel zur Bestimmung, ob bestimmte Proteine ​​einzigartigen Positions-Lokalisierungen Ausstellung in 3D-Kultur bietet, kann man folgern, und testen Protein Funktion und Regulation im Hinblick auf regionale Lokalisation innerhalb der Neurosphäre werden, und, was vielleicht am wichtigsten , bietet die Positionsdaten benötigt, um die Auswirkungen der 3D-Topographie und zellulären Positionierung auf NPC Schicksal in 3D-Kultur zu studieren.

* Alle anderen Standard-chemischen Reagenzien im Protokoll aufgelistet sind von Sigma-Aldrich. Alle anderen Standard-Zellkultur-Reagenzien sind von Invitrogen.

localizing protein in 3d neural stem cell culture: a hybrid visualization methodology

Die Bedeutung der 3-dimensional (3D) Topographie beeinflussen neuralen Stamm-und Progenitorzellen (NPC)-Phänotyp ist allgemein anerkannt, aber schwierig zu studieren. Wenn losgelöst von embryonalen oder postnatale Gehirn, werden einzelne NPCs in Suspension vermehren zu Neurosphären bilden. Tochterzellen innerhalb dieser Kulturen spontan annehmen deutliche Entwicklungsstörungen Linien (Neuronen, Oligodendrozyten und Astrozyten) im Laufe der Expansion trotz der gleichen extrazellulären Milieu ausgesetzt. Diese Entwicklung rekapituliert viele der Stadien beobachtet im Laufe der Neurogenese und Gliogenese in postnatalen Gehirn und wird häufig verwendet, um grundlegende NPC Biologie in einer kontrollierten Umgebung zu studieren. Die Beurteilung der vollen Auswirkungen der 3D-Topographie und zellulären Positionierung innerhalb dieser Kulturen auf NPC Schicksal ist jedoch schwierig. Zur Lokalisierung von Target-Proteinen und identifizieren NPC Linien Immunzytochemie, muss frei schwebenden Neurosphären auf einem Substrat beschichtet werden oder seriell geschnitten. Diese Verarbeitung ist erforderlich, um gleichwertige Zellpermeabilisierung und Antikörper-Zugang im gesamten Gebiet zu gewährleisten. Als Ergebnis können 2D epifluoreszenten Bilder von Gefrierschnitten oder konfokale 3D-Rekonstruktionen von Z-Stapeln nur stellen räumliche Informationen über Zell-Position innerhalb diskreten physischen oder digitalen 3D-Scheiben und nicht visualisieren zelluläre Position im intakten Bereich. Hier, in der die Topographie des Neurosphäre Kultur bekräftigen und erlauben räumliche Analyse der Proteinexpression in der gesamten Kultur präsentieren wir ein Protokoll für die Isolierung, Expansion und serielle Schnitte von postnatalen Hippocampus Neurosphären für epifluoreszenten oder konfokalen Immundetektion der Zielproteine. Connexin29 (Cx29) wird als Beispiel analysiert. Anschließend mit einem Hybrid aus Grafik-Bearbeitung und 3D-Modellierung Software rigoros angewendet, um biologische Detail zu halten, beschreiben wir, wie der Zusammenbau des 3D strukturelle Positionierung der Bilder und digital map markierten Zellen innerhalb der gesamten Neurosphäre. Diese Methode ermöglicht die Visualisierung und Analyse der zellulären Position von Target-Proteinen und Zellen über die gesamte 3D-Topographie und Kultur wird eine detailliertere Analyse der räumlichen Beziehungen zwischen den Zellen im Laufe der Neurogenese und Gliogenese in vitro zu erleichtern. Beide Imbeault und Valenzuela gleichermaßen beigetragen und sollte als gemeinsame Erstautoren werden.

Watch this Scientific Journal Video about Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology at JoVE.com

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

Hier beschreiben wir, wie zu produzieren, zu erweitern und immunolabel postnatalen Hippocampus neuronale Vorläuferzellen (NPCs) im dreidimensionalen (3D-) Kultur. Als nächstes mit Hybrid-Visualisierungs-Technologien, demonstrieren wir, wie digitale Bilder von immunolabelled Kryoschnitten verwendet werden, um zu rekonstruieren und Karte der räumlichen Lage der immunpositive Zellen über die gesamte 3D-Neurosphäre werden.

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 ...

localizing-protein-3d-neural-stem-cell-culture-hybrid-visualization

Research

JoVE Journal

Neuroscience

12.6K Aufrufe.  University of Ottawa. Hier beschreiben wir, wie zu produzieren, zu erweitern und immunolabel postnatalen Hippocampus neuronale Vorläuferzellen (NPCs) im dreidimensionalen (3D-) Kultur. Als nächstes mit Hybrid-Visualisierungs-Technologien, demonstrieren wir, wie digitale Bilder von immunolabelled Kryoschnitten verwendet werden, um zu rekonstruieren und Karte der räumlichen Lage der immunpositive Zellen über die gesamte 3D-Neurosphäre werden.

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

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

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Wachstum und Differenzierung von adulten Hippocampus Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

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.

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 ...

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). ...

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. ...

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

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Einfache Erstellung einer High-Yield-Kultur der induzierten Neuronen aus Menschenhaut Erwachsenen Fibroblasten

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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

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