Die Unterstützung und Beratung von Markus Pajtler und Robert Bohrer waren von unschätzbarem Wert für die Visualisierung dieses Protokolls.

Die Autoren stellen fest, dass Tierversuche in Übereinstimmung mit der Europäischen Gemeinschaften Richtlinie (86/609/EWG) durchgeführt wurden, nach den Richtlinien der NIH zur Pflege und Verwendung von Tieren für experimentelle Verfahren und den Regularien der Institutional Tiere gesetzt Pflege und Nutzung Committee (IACUC) an der Universität Duisburg-Essen (Deutschland). Teil 1: Einrichten (nicht auf Video gezeigt) Vorbereitung von Materialien und Lösungen (vor der Einnahme der Eier aus dem Inkubator) <ul><li> Eier haben, um in einem befeuchteten Inkubator für 11 Tage bei 37 ° C (100 ° F). </li><li> 70% Ethanol-Spray, saubere Platten, präzise sauberen Pinzette, 4 ° C (40 ° F) kalte sterile DMEM auf Eis und 60 ml sterilen Spritzen </li><li> 50 ml Sterile Corning Rohre müssen mit 4 halbgefüllten ° C kalten DMEM der mazeriert Embryonen bei einem Verhältnis von 1:1 (mazeriert Masse: DMEM) mischen. Sobald die Eier außerhalb des Inkubators Dissektion der Hühnerembryonen ergriffen hat, um gestartet werden. </li><li> Sterilisieren Dissektion Werkzeuge im Autoklaven oder am Tag der Dissektion, tauchen die Werkzeuge in 70% Ethanol für 20 Minuten. </li></ul> Teil 2: Präparation der Hühnerembryonen (gezeigt auf Video) <ol><li> Wet den bebrüteten Eiern mit 70% Ethanol-Spray für mindestens 30 Sekunden und trocknen Sie sie sorgfältig mit einem sauberen weichen Papiertüchern zwar nicht Schütteln der Eier nicht übermäßig. Hinweis: Wir empfehlen nicht sezieren mehr als 60 Hühnerembryonen gleichzeitig. </li><li> Öffnen Sie die Eier vorsichtig von schneidigen gegen eine scharfe Kante und nehmen Sie den Embryo mit Vorsicht nicht die Zerstörung der Dottersack oder der Hühnerembryo sich. </li><li> Suchen Sie nach dem Beginn der Nabelschnur schnell und öffnen Sie die klare Verpackung ohne Zerstörung der Dottersack oder kleinen Gefäßen. Dann sezieren die Nabelschnur mit präzisen sauberen Pinzette. </li><li> Nehmen Sie den Embryo aus dem Dottersack. Hinweis: Die Embryonen sollten schnell enthauptet werden (nicht im Video gezeigt). </li><li> Sammeln Sie die Embryonen in Minimal Essential Medium bei 4 ° C. </li></ol> Teil 3: Mazeration der Hühnerembryonen <ol><li> Nehmen Sie den Kolben aus einer 60 mL sterile Spritze aufziehen. </li><li> Transfer ca. 10 Embryonen in einer 60 ml Spritze. </li><li> Setzen Sie die Kolben vorsichtig zurück in die Spritze, um nicht zu riskieren mazeriert Material. </li><li> Mazerat durch Herunterdrücken des Kolbens. </li><li> Sammeln Sie die mazeriert Masse in die vorbereitete Corning Rohre mit DMEM bei einem Verhältnis von 1:1 (mazeriert Masse: DMEM) zur Hälfte gefüllt. Mit 10 Embryonen ein Volumen von etwa 25 mL hergestellt wird. </li><li> Legen Sie die Mischung auf einem Schüttler für 45 min bei 4 ° C. </li></ol> Teil 4: Zentrifugation der Hühnerembryo - DMEM Suspension <ol><li> Übertragen Sie die Hühnerembryo - DMEM Suspension in die Zentrifugenröhrchen. </li><li> Add sterile Hyaluronidase in einer Konzentration von 1 mg pro 25 mg des Embryos. </li><li> Tare die Zentrifugenröhrchen sehr genau. Hinweis: Dieser Schritt sollte mit Vorsicht durchgeführt werden, wie Zentrifugation bei sehr hohen g-force und Schäden materieller oder Zentrifuge kann auftreten, wenn mit unausgeglichenen Rohren ausgeführt wird. Mit einer hohen Genauigkeit Waage nach zu den drei Nachkommastellen für alle Röhren durch eine Anpassung der fehlende Betrag mit DMEM empfangen werden soll. </li><li> Reduzieren Sie die Temperatur der Zentrifuge auf 4 ° C. Hinweis: Auch die Zentrifugation Rotor sollte in einer Kühlkammer oder Kühlschrank über Nacht aufbewahrt werden. </li><li> Centrifuge 6 Stunden mindestens für 180.000 xgx h. Hinweis: Für bessere Ergebnisse in Bezug auf die Trennung der einzelnen Phasen zu verwenden 266,000 xgx h. Wenn möglich, verwenden Sie das Vakuum Anpassung. </li></ol> Teil 5: Filtration des Hühnerembryo-Extrakt Nach der Zentrifugation können drei Phasen zu beachten: Die obere schmuddeligen flüssigen Phase mit Fett-Fragmente, die klare flüssige Phase in der Mitte und das Pellet am Boden der Zentrifugenröhrchen. Hinweis: Schütteln oder rege Bewegung der zentrifugierten Mischung muss strikt zu vermeiden, da dies das Ergebnis der Zentrifugationsschritt durch den Verlust des klaren zentralen flüssigen Phase zu zerstören. Hinweis: Alle folgenden Schritte wurden unter sterilen Bedingungen mit einem laminaren Luftstrom Bank durchgeführt werden: <ol><li> Pipette die klare flüssige Phase in ein Filtersystem mit Poren von 0,45 um. Dann auf dem angeschlossenen Vakuumpumpe einschalten. Hinweis: Berühren Sie nicht die Pellet am Boden. Dies würde zu einer Verunreinigung des Extraktes und ein Verstopfen des Filters Systems führen. </li><li> Übertragen Sie die Pre-Extrakt in einen Filter mit Poren von 0,22 um und schalten Sie die angeschlossenen Vakuumpumpe wieder. </li><li> Aliquot der erzielten Hühnerembryo-Extraktin sterile 15 mL Corning Röhren. </li><li> Schnell speichern Sie die Aliquots bei -80 ° C (-62 ° F). Hinweis: Die CEE dauern bis zu zwei Jahren, wenn bei -80 ° C. </li></ol>

<ol>
	<li>Bronner-Fraser, M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+reveals+multipotency+of+some+avian+neural+crest+cells.">Cell lineage analysis reveals multipotency of some avian neural crest cells.</a> Nature. 335, 161-164 (1988).</li><li>Stemple, D. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Rao, M. S., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+controlled+in+vitro+differentiation+of+murine+multipotent+neural+crest+stem+cells.">Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.</a> J Neurobiol. 32, 722-746 (1997).</li><li>Paratore, C., Goerich, D. E., Suter, U., Wegner, M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+glial+fate+acquisition+of+neural+crest+cells+are+regulated+by+an+interplay+between+the+transcription+factor+Sox10+and+extrinsic+combinatorial+signaling.">Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling.</a> Cell. 128, 3949-3961 (2001).</li><li>Kim, J., Lo, L., Dormand, E., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX10+maintains+multipotency+and+inhibits+neuronal+differentiation+of+neural+crest+stem+cells.">SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells.</a> Neuron. 38, 17-31 (2003).</li><li>Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao, M., Shah, N. M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+determination+and+the+control+of+neuronal+identity+in+the+neural+crest.">Cell lineage determination and the control of neuronal identity in the neural crest.</a> Review Cold Spring Harb Symp Quant Biol. 62, 493-504 (1997).</li><li>Dorsky, R. I., Moon, R. T., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+and+cell+fate+specification+in+premigratory+neural+crest.">Environmental signals and cell fate specification in premigratory neural crest.</a> Review Bioessays. 22, 708-716 (2000).</li><li>Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+lineage+specification+in+the+neural+crest.">Factors controlling lineage specification in the neural crest.</a> Int Rev Cytol. 197, 1-33 (2000).</li><li>Dupin, E., Real, C., Ledouarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+crest+stem+cells:+control+of+neural+crest+cell+fate+and+plasticity+by+endothelin-3.">The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3.</a> Int Rev Cytol. 73, 533-545 (2001).</li><li>Maurer, J., Fuchs, S., J&#228;ger, R., Kurz, B., Sommer, L., Schorle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+controlled+differentiation+of+neural+crest+stem+cell+lines+using+conditional+transgenesis.">Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis.</a> Differentiation. 75, 890-891 (2007).</li><li>Wu, Y. Y., Mujtaba, T., Rao, M. 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+of+stem+and+precursor+cells+from+fetal+tissue.">Isolation of stem and precursor cells from fetal tissue.</a> Methods Mol Biol. 198, 29-40 (2002).</li></ol>

Dieses Protokoll basiert auf Modifikationen der Verfahren, die in den letzten 10,11 beschrieben worden sind, basiert. Zusätzlich zu früheren Veröffentlichungen haben wir hier zeigen die einzelnen unterschiedlichen Schritte des Prozesses mehr ausgeweitet. Dies unterstützt den Experimentator mit den wichtigsten Daten sichern richtige Verfahren und zuverlässige Ergebnisse. Ferner ist, wie die Zerlegung Prozess ist ein wesentlicher Bestandteil der CEE-Extraktion beschreiben wir das Verfahren der Aufnahme der...

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<td>11 days incubated chicken eggs</td>
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<td>S&ouml;rries-Trockels Vermehrungszucht GmbH &amp; Co.KG</td>
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<td>Alcoholic disinfection spray</td>
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<td>Sch&uuml;lke &amp; Mayr GmbH</td>
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<td>DMEMGlutamax</td>
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<td>GIBCO Invitrogen</td>
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<td>Syringe sterile (60 ml)</td>
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<td>BD Biosciences</td>
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<td>Tubes sterile (50; 15 ml)</td>
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<td>Greiner Bio-One GmbH</td>
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<td>Pipet tips sterile</td>
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<td>Starlab GmbH</td>
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<td>Hyaluronidase typeIV-S</td>
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<td>Sigma-Aldrich</td>
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<td>Milipore stericups sterile (0.45; 0.22 &mu;l)</td>
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<td>Fisher Scientific AG</td>
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<td>Pipettes sterile (25; 10; 5 ml)</td>
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<td>Greiner Bio-One GmbH</td>
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<td>Centrifuge L5-50</td>
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<td>Rotor type Ti-45</td>
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production of chick embryo extract for the cultivation of murine neural crest stem cells

Die Neuralleiste ergibt sich aus der Neuro-Ektoderm während der Embryogenese und besteht nur vorübergehend. Frühe Experimente bereits geprüften pluripotenten Vorläuferzellen zu einem integralen Bestandteil der Neuralleiste 1 sein. Phänotypisch sind Neuralleiste Stammzellen (NCSC) durch die simultane Expression p75 (low-affine nerve growth factor receptor, LNGFR) und SOX10 während ihrer Wanderung von der Neuralleiste 2,3,4,5 definiert. Diese Vorläuferzellen können in glatten Muskelzellen, chromaffinen Zellen, Neuronen und Gliazellen, sowie Melanozyten, Knorpel und Knochen 6,7,8,9 unterscheiden. Zu kultivieren NCSC in vitro, eine besondere Neuralleiste Stammzellen Medium (NCSCM) ist 10 erforderlich. Der komplexeste Teil des NCSCM ist die Herstellung von Hühnerembryo-Extrakt (CEE) vertreten eine wesentliche Quelle von Wachstumsfaktoren für die NCSC sowie für andere Arten von neuronalen Explantaten. Andere NCSCM Zutaten neben CEE sind im Handel erhältlich. Producing CCE mit Labor-Grundausstattung ist es von großer Bedeutung, um über die schwierigen Details wie die Isolierung, Mazeration, Zentrifugation und Filtration Prozesse kennen. In diesem Protokoll beschreiben wir genauer Techniken, um eine maximale Menge an reinem und hochwertigem CEE produzieren.

Watch this Scientific Journal Video about Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells at JoVE.com

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

Um Neuralleiste Stammzellen (NCSC) in vitro zu kultivieren, ist ein spezielles Medium (NCSCM) erforderlich. Wesentlicher Bestandteil der NCSCM ist Hühnerembryo-Extrakt (CEE). Wir beschreiben hier genaue Techniken, um eine maximale Menge an reinem und hochwertigem CEE, einschließlich Details wie die Isolierung, Mazeration, Zentrifugation und Filtration zu produzieren.

Herstellung von Hühnerembryo-Extrakt für die Kultivierung von Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

production-chick-embryo-extract-for-cultivation-murine-neural-crest

Research

JoVE Journal

Neuroscience

12.6K Aufrufe.  University Children's Hospital Essen. Um Neuralleiste Stammzellen (NCSC) in vitro zu kultivieren, ist ein spezielles Medium (NCSCM) erforderlich. Wesentlicher Bestandteil der NCSCM ist Hühnerembryo-Extrakt (CEE). Wir beschreiben hier genaue Techniken, um eine maximale Menge an reinem und hochwertigem CEE, einschließlich Details wie die Isolierung, Mazeration, Zentrifugation und Filtration zu produzieren.

Serie: Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Isolation und Kultur von Neural Crest Stem Cells aus menschlichen Haarfollikeln

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Forschung

Neurowissenschaften

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 ...

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

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

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

Feeder freie Ableitung von Neural Crest Vorläuferzellen aus menschlichen pluripotenten Stammzellen

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

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 ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Der Nachweis von 5-Hydroxymethylcytosin in neuralen Stammzellen und Gehirnen von Mäusen

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Organotypische Kulturen des adulten humanen Kortex als Ex-vivo-Modell für die Transplantation und Validierung humaner Stammzellen

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

Gewinnung enterischer Neuronen aus humanen pluripotenten Stammzellen

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

Autor im Rampenlicht: Robuste Kultur menschlicher enterischer Neuronen aus humanen pluripotenten Stammzellen

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is presented.

Differenzierung der Abstammungslinien des enterischen Nervensystems von humanen pluripotenten Stammzellen

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls ...