HD054347 und NS063997-01 bis VLS: Diese Arbeit wurde zum Teil durch die National Institutes of Health unterstützt. Diese Arbeit wurde zum Teil auch durch den Empire State Stem Cell Fonds durch die New York State Department of Health Contract # C024324 zu VLS unterstützt. Die darin geäusserten Meinungen sind hier ausschließlich die des Autors und spiegeln nicht unbedingt die des Empire State Stem Cell Board, das New York State Department of Health, oder des Staates New York. VLS ist ein Doris Duke Clinical Scientist Developmental Preisträgerin. Wir danken auch Professor Timothy Vartanian für seine Gabe der Anti-O1, Anti-O4-Antikörper.

1. Herstellung von Lösungen und Materialien für die Präparation und Pflege von neuralen Stammzellen Kultur <ol><li> Bereiten 100ml Dissektion Medium (KNOCKOUT DMEM/F12, Invitogen) vor der Zeit und in den Kühlschrank. </li><li> Prepare100 ml Kulturmedium (Stem Pro NSC SFM, Invitrogen) und halten bei 37 ° C in einem Wasserbad. </li><li> Bereiten Zell-Einfriermedium (KNOCKOUT DMEM/F12 +10% FBS + 5% DMSO) für die langfristige Kryokonservierung von Zellen. </li><li> Wenn gewünscht, bereiten 4% Paraformaldehyd (PFA) für Gewebefixierung. </li><li> Sterile, autoklaviert Pinzette und Skalpell Klingen mit Griffe sind für die Präparation verwendet. </li><li> Beiseite einer Pipette Pistole, 10 ml Transferpipetten und 40 uM Zellsieb (BD Falcon 352340) für Dissoziation. </li><li> Beiseite mehrere 10 cm Kulturschalen (BD) für die Präparation, 50 ml Zentrifugenröhrchen (BD) für die Dissoziation, 1,5 ml Röhrchen für die Gewebe-Lagerung und gefrorene Röhrchen (BD) für das Einfrieren von Zellen. </li></ol> 2. Isolierung humaner neuraler Stammzellen aus dem menschlichen fetalen Gehirns <ol><li> Die Ernte der lebensfähiges Gewebe sofort nach Beendigung des Fötus ist entscheidend für den Erfolg des Verfahrens. Für elektive Prozeduren können Timing, um Proben zu erhalten im Voraus gebucht werden, um so Zeit nach fetalen Tod zu minimieren. Die Produkte der Empfängnis sind innerhalb von 2 Stunden nach dem Eingriff geerntet, sondern im Idealfall auf elektiven Eingriffen innerhalb weniger Minuten erreicht werden. Die fetalen Gewebe oft fragmentiert sind. Im allgemeinen sind jedoch ein wesentlicher Teil des Gehirns bleibt für visuelle Identifizierung. Einschränkungen von Gestationsalter (GA) werden von gesetzlichen Vorschriften bestimmt, sondern wurden unter Verwendung dieses Protokolls zwischen 18-22 Wochen GA. </li><li> Fetal Gehirn ist in einer 10 ml Petrischale mit eiskaltem KNOCKOUT DMEM/F12 Lösung gegeben. Identifizieren verschiedenen Teilen des Kortex von anatomischen Landmarken. Grenzen für die Frontal-und parieto-occipital Kortex richtet sich nach den hochgerechneten Schnittpunkt der zentralen Sulcus und Sylvi-Fissur. Dissect Gewebe aus dem frontalen Kortex anterior des Sulcus centralis und entlang der Grenze von der Sylvi-Fissur mit chirurgische Messer, achten Sie darauf, halten den Ventrikel intakt und unbeschädigt sein. </li><li> Entfernen Sie alle verbleibenden Blut und Meningen aus dem abgetrennten Block von frontalen Kortex. Wenn die Probe von ausreichender Qualität, ist es ideal, um den Block in mehrere kleinere Proben für verschiedene Zwecke zu sezieren: Sektionen (in 4% Paraformaldehyd, PFA) und Protein / mRNA-Assays (schnell in -80 ° C gefroren). </li><li> Übertragen Sie die ausgewählten Gehirn blockieren, um ein 50 ml Zentrifugenröhrchen, und fügen Sie eiskalten KNOCKOUT DMEM/F12 Lösung bei etwa 3-mal des Gewebes der Lautstärke. Vorsichtig distanzieren das Gewebe durch mechanische Pipettieren mit einer 10 ml Vollpipette, bis alle Gewebe fragmentiert wird (in der Regel 20-30 mal) und dann filtern Sie die Zellen durch eine 40 uM Zellsieb (BD Falcon 352340) um Einzel-oder in der Nähe einzelnen Zelle zu erhalten Suspension. </li><li> Centrifuge die Zellsuspension bei 2000 rpm und Raumtemperatur für 5 min, Zellpellet in 10 ml frisches, warmes Kulturmedium (Stem Pro NSC SFM), und zählen Sie die Anzahl der Zellen mit einer Zählkammer. </li><li> 5 ml warmem Kulturmedium in je 25 cm 2 Kulturflaschen und Transfer 2x10 6 Zellen in jeden Kolben. Kulturen sind in einem 37 ° C / 5% CO 2-Inkubator für 1 Woche vor der Analyse. Ändern Sie die Hälfte des Mediums einmal pro Woche für weitere Kulturen oder Experimente. </li></ol> 3. Manipulaton neuraler Stammzellen für die weitere Charakterisierung oder Experimente <ol><li> Neurosphären in der Regel in 1 bis 2 Wochen bilden unter den empfohlenen Kulturbedingungen mit NSC Durchmesser im Bereich zwischen 200 und 400 um. Neurosphären in diesem Stadium kann mit 0,2 g / L EDTA in Calcium-und Magnesium freie Hanks Medium (Hanks) bei 37 ° C dissoziiert für 15 min auf einzelne Zellen zu erhalten. Cells Suspension bei 2000 rpm, in frischen Hanks gespült gesponnen, und replattiert in warmen Nährboden für Subkultur. </li><li> Zur Einleitung Differenzierung werden dissoziierten Zellen auf poly-D-lysine/laminin 1 beschichtete Deckgläser in einer Dichte von 1x10 5 Zellen pro Deckglas (24mmX24mm) ausplattiert. Oligodendrozyten-Differenzierung wird durch die Aufrechterhaltung der Zellen in KNOCKOUT DMEM/F12 (Invitrogen, Main, MD) +2% B27 (50X, Invitrogen, Main, MD) +10 ng / ml bFGF 100 ng / ml SHH + 10ng/ml PDGF-AA für erreicht 2 Tage, dann Umstellung auf das gleiche Medium ohne Wachstumsfaktoren für weitere 5 Tage. Neuronale Differenzierung wird durch das Aufrechterhalten Zellen in KNOCKOUT DMEM/F12 +2% B27 (50x) für 7 Tage erreicht. Astrocyte Differenzierung wird durch Kultivieren von Zellen in KNOCKOUT DMEM/F12 +1% FBS für eine Woche getan. </li><li> Die Transfektion von neuronalen Stammzellen mit Gene können mit dissoziierten Zellen aus dem vorgeformten Neurosphären durchgeführt werden. Hier haben wir gezeigt, dass die Stammzellen mit EGFP-C1 durch Elektroporation transfiziert. Die Elektroporation von EGFP-C1-Konstrukt wurde unter Verwendung von amaxa Nucleofector Kits für Mouse Neural Stem Cells (VPG-1004) mit amaxa Nucleofector Device (Lonza AAD-1001), nachdem das Unternehmen die Anweisung. Kurz gesagt, wurden 5 ug DNA mit 1 x 10 6 Zellen mit 100 ul Transfektion Medium gemischt und nach Puls Elektroporation wurden die Zellen in der neuralen Stammzellen erhalten Medium für weitere Kultur resuspendiert. Die Differenzierung der transfizierten Zellen wurden mit der Dissoziation von Neurosphären 3-4 Tage nach der Elektroporation verarbeitet, nach dem gleichen Verfahren in Schritt 3.2 beschrieben. </li></ol> 4. Freezing neuralen Stammzellen und Subkulturen <ol><li> Dissoziiert Zellsuspensionen werden zentrifugiert und in Einfriermedium mit einer Konzentration von 1x10 7 Zellen / Flasche / ml. Langsam frieren die Zellen in -20 ° C, -80 ° C dann in flüssigen Stickstoff Transfer für lange Lagerung. </li><li> Die Zellen werden schnell mit 37 ° C aufgetaut Wasserbad gewaschen und in erwärmt DMEM/F12 +10% Serum für eine Wäsche, zentrifugiert, um das Einfrieren Medium zu entfernen, und erneut in die erwärmte Kulturmedium. </li></ol> 5. Repräsentative Ergebnisse: Neurale Stammzellen von einem normalen Fötus mit 18 Wochen Schwangerschaftsdauer wurden nach dem beschriebenen Verfahren und Neurosphären nach einer Woche mit runden, glatten Rändern und ziemlich homogen Größe (Abb. 2a) zu sehen ist, kultiviert. Diese Neurosphären mit EGFP-C1 oder anderen Konstrukten transfiziert und anschließend unter Fluoreszenz-Mikroskopie (Abb.2b,). Gegründet Neurosphären wurden dann dissoziiert mit EDTA und verchromt als dispergierte Zellen auf beschichteten Deckgläsern. Zellen, die unter den jeweiligen Protokollen unterschieden wurden mit 4% parafamaldehyde fixiert und gefärbt mit verschiedenen Zelltyp spezifischen Markern. Multipotentiality ist mit der Expression von Markern bezeichnend für Neuronen (Fig2C, D, Rhodamin) Astrozyten (Fig2E, F, Rhodamin) und Oligodendrozyten (Fig2G, H, Rhodamin) beobachtet. Die Zellen nicht erworben Elektroporation von EGFP wurden auch in verschiedenen Zelltypen differenzieren und gefärbt mit verschiedenen Zell-spezifische Marker. Multipotentiality ist mit der Expression von Markern bezeichnend für Neuronen (Fig3A, B, Fluorescein) Astrozyten (Fig3C, Rhodamin und Fig3D, Fluorescein) und Oligodendrozyten (Fig3E, F, Rhodamin) beobachtet. <img src="/files/ftp_upload/2681/2681fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Schematische Darstellung des experimentellen Verfahren zur neuralen Stammzellen aus gebrauchten menschlichen fetalen Gehirns zu isolieren <img src="/files/ftp_upload/2681/2681fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Undifferenzierte und differenzierte menschliche Nervenzellen in vitro propagiert. (A) Neurosphären unter Phasenkontrast-Mikroskopie gezeigt demonstrieren glatte, runde Grenzen und schnelles Wachstum nach der Kultur seit über 1 Woche. (B) Einführung der verschiedenen Plasmide und Konstrukte können durch Transfektion erreicht werden. Drei Tage nach der EGFP-C1-Transfektion, zeigen mehrere Zellen die Expression des grün fluoreszierenden Proteins, wie unter Fluorescein Immunfärbung und Fluoreszenzmikroskopie zu sehen. EGFP-C1 transfizierten Neurosphären werden dissoziiert und differenziert unter verschiedenen Bedingungen zu Neuronen (C, D), Astrozyten (E, F) und Oligodendrozyten (G, H) und sehen unter Rhodaminfluoreszenz. Gleichzeitig sind transfizierten EGFP-positiven Zellen unter Fluorescein-Fluoreszenz gezeigt. Transfizierten Zellen (weiße Pfeilspitzen) sind zu unterscheiden von nicht transfizierten Zellen (weiße Pfeile). Die Zellkerne sind mit Hoechst33342 gefärbt. Scale-Bars sind 200 pm für A, 100 pm für B und 25 um für CH. <img src="/files/ftp_upload/2681/2681fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Neurosphären ohne Transfektion sind dissoziiert und differenziert unter verschiedenen Bedingungen in Neuronen (A, B, Fluorescein), Astrozyten (C, Rhodamin, D, Fluorescein) und Oligodendrozyten (E, F, Rhodamin). Die Zellkerne sind mit Hoechst33342 gefärbt. Scale-Bars sind 25 mu m für AF.

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Es gibt verschiedene Ansätze zur Kultur frischem Gewebe und Herstellung von menschlichen Zelllinien. Historisch gesehen hat frischem Gewebe geerntet und sofort in verschiedenen Zelltypen im zentralen Nervensystem zu erzeugen kultiviert. Dieser Ansatz ist jedoch klar durch die Anzahl der Proben, die erhalten-die im Falle der menschlichen Proben, ist in der Regel recht klein werden kann, begrenzt. Angesichts der minimalen Grad an Manipulation, bieten frisch gezüchteten Nervenzellen den zuverlässigsten experimentelles S...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> KNOCKOUT DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td> Dissoziation Medium </td></tr><tr><td> Stem Pro NSC SFM </td><td> Invitrogen </td><td> A10509-01 </td><td> Nährboden </td></tr><tr><td> Fetal Bovine Serum </td><td> Invitrogen </td><td> 10091-148 </td><td> Gefrorenes Medium </td></tr><tr><td> Hanks-Lösung (-Ca 2 +,-Mg 2 +) </td><td> Invitrogen </td><td> 14175-095 </td><td> Dissoziation Medium </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D2650 </td><td> Gefrorenes Medium </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> 431788 </td><td> Dissoziation Medium </td></tr><tr><td> Paraformaldehyd </td><td> Sigma-Aldrich </td><td> 158127 </td><td> Fixation Lösung </td></tr><tr><td> bFGF </td><td> R &amp; D </td><td> 234-FSE </td><td> Differenzierungsmedium </td></tr><tr><td> SHH </td><td> R &amp; D </td><td> 1845-SH </td><td> Differenzierungsmedium </td></tr><tr><td> PDGF-AA </td><td> R &amp; D </td><td> 221-AA </td><td> Differenzierungsmedium </td></tr><tr><td> B27 </td><td> Invitrogen </td><td> 17504-044 </td><td> Differenzierungsmedium </td></tr><tr><td> Maus Anti-MAP2 </td><td> Sigma-Aldrich </td><td> M2320 </td><td> 1:200 </td></tr><tr><td> Kaninchen Anti-DCX </td><td> Zelluläre Signale </td><td> 4604s </td><td> 1:200 </td></tr><tr><td> Kaninchen Anti-GFAP </td><td> DAKO </td><td> Z0334 </td><td> 1:200 </td></tr><tr><td> Kaninchen Anti-S100B </td><td> DAKO </td><td> Z0311 </td><td> 1:200 </td></tr><tr><td> Kaninchen Anti-O1 </td><td> Geschenke von Professor Timothy Vartanian * </td><td></td><td> 01.50 </td></tr><tr><td> Kaninchen Anti-O4 </td><td> Geschenke von Professor Timothy Vartanian * </td><td></td><td> 01.50 </td></tr><tr><td> 40 um Zellsieb </td><td> BD Falcon </td><td> 352340 </td><td></td></tr></tbody></table> * Timothy Vartanian, MD, PhD, Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, USA

generation of neural stem cells from discarded human fetal cortical tissue

Neurale Stammzellen (NSCs) entlang der ventrikulären Zone Neuroepithel wohnen während der Entwicklung der kortikalen Platte. Diese frühen Vorfahren die zu weiteren Zwischen-Vorläuferzellen und später die verschiedenen neuronalen und glialen Zell-Subtypen, dass die Großhirnrinde bilden. Die Fähigkeit, zu generieren und zu erweitern menschlichen NSCs (sog. Neurosphären) aus gebrauchten normalen fetalen Gewebe bietet ein Mittel, mit denen direkt zu untersuchen, um die funktionalen Aspekte der normalen menschlichen NSC Entwicklung 1-5. Dieser Ansatz kann auch auf die Generation der NSCs aus bekannten neurologischen Erkrankungen gerichtet sein, wodurch sich die Möglichkeit, Krankheitsprozesse, die Vorläuferzellen Proliferation, Migration und Differenzierung 6-9 verändern zu identifizieren. Wir haben auf die Identifizierung pathologischer Mechanismen im menschlichen Down-Syndrom NSCs, die zur beschleunigten Alzheimer-Phänotyp 10,11 beitragen könnten konzentriert. Weder in vivo noch in vitro-Maus-Modelle replizieren kann die gleiche Repertoire von Genen auf dem menschlichen Chromosom 21 befindet. Hier verwenden wir eine einfache und zuverlässige Methode zur Isolierung Down-Syndrom NSCs aus abgetriebenen menschlichen Föten Kortex und wachsen sie in der Kultur. Die Methodik bietet spezifische Aspekte der Ernte der Gewebe, Dissektion mit begrenzten anatomischen Landmarken, Zellsortierung, Beschichtungs-und Passagieren des menschlichen NSCs. Wir bieten auch einige grundlegende Protokolle zur Induktion Differenzierung von humanen NSCs in selektiver Zell-Subtypen.

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Eine einfache und zuverlässige Methode zur Isolierung und Kultivierung von neuralen Stammzellen aus gebrauchten menschlichen fetalen kortikalen Gewebe wird beschrieben. Kulturen von bekannten menschlichen neurologischen Erkrankungen abgeleitet werden können für die Charakterisierung von pathologischen zellulären und molekularen Prozesse eingesetzt werden, sowie eine Plattform bieten, um pharmakologische Wirksamkeit zu beurteilen.

Generierung von neuralen Stammzellen aus menschlichen Föten Ausrangierte Rindengewebe

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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15.4K Aufrufe.  Beth Israel Deaconess Medical Center. Eine einfache und zuverlässige Methode zur Isolierung und Kultivierung von neuralen Stammzellen aus gebrauchten menschlichen fetalen kortikalen Gewebe wird beschrieben. Kulturen von bekannten menschlichen neurologischen Erkrankungen abgeleitet werden können für die Charakterisierung von pathologischen zellulären und molekularen Prozesse eingesetzt werden, sowie eine Plattform bieten, um pharmakologische Wirksamkeit zu beurteilen.

Serie: Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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). ...

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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 ...

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 ...

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

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

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

Ein cGMP-Verfahren zur Erweiterung anwendbar Aggregate von humanen neuralen Stamm-und Vorläuferzellen, abgeleitet von pluripotenten Stammzellen oder fetalen Hirngewebe

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

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

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

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

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 ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modellierung Astrozytom Pathogenese<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt; Verwenden kortikale Astrozyten oder neuralen Stammzellen aus dem Bedingten, gentechnisch veränderte Mäuse

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

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 ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Gleichzeitige Aufnahmen der kortikalen lokales Feld Potentiale, Elektrokardiogramm, Elektromyogramm und Atemrhythmus aus einem frei beweglichen Ratte

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...