Diese Arbeit wurde von Merit Bewertung Zuschuss (NEUD-004-07F) von der Veterans Administration (SP) unterstützt.

1. Erweiterung des menschlichen NPCs (Abbildung 1) <ol><li> Revive die Tiefkühllagerung von menschlichen NPCs aus fötalen Gehirns (Lonza Walkersville, MD, USA) und Kultur sie in Suspension in T-75-Flaschen als Neurosphären (Abbildung 1A) in Neurobasalmedium enthält Proliferation Ergänzungen, EGF (10 ng / ml) und FGF (10 ng / ml). </li><li> Nach 3 Tagen in Kultur übertragen die Neurosphären in ein 15 ml Zentrifugenrohr und bei 500 rpm für 5 min. </li><li> Überstand verwerfen und hinterließ ~ 100 ul Medium über dem Zellpellet und Überführung in ein Eppendorf-Röhrchen. A 100 ul Zellpellet ist genug, um in zwei T-75-Flaschen aufgeteilt. Wenn weniger, reduzieren Sie die Anzahl der Fläschchen, als neuroprogenitor Zellen nicht zu vermehren, wenn aufgeteilt dünn. </li><li> Verreiben des Pellets mit einer Spitze 200 ul 50x, während die 200 ul Spitze gegen den Boden des Röhrchens. Teilen Sie die Zellsuspension in zwei gleiche Teile und fügen Sie sie in zwei T-75-Flaschen (1-2 split), eine für die FortsetzungExpansion und eine weitere Differenzierung. </li><li> Fahren Sie mit der Kultur der neuroprogenitor Zellen für die Erweiterung (erste Kolben), indem das Medium alle 4-5 Tage, bis die Neurosphären ihrer ursprünglichen Größe von 300-500 um zu erreichen. Ändern Medium durch Zentrifugation (500 rpm, 5 min), entsorgen Sie das alte Medium und neue Medium. </li></ol> 2. Differenzierung von humanen NPCs in ein Neuron reiche Kultur (Abbildung 2) <ol><li> Für die Differenzierung von Zellen in eine neuroprogenitor Neuron reiche Kultur, legen Sie eine einzelne Deckglas in jede Vertiefung einer 24-Well-Platte und Mantel mit 100 ug / ml Poly-L-Lysin durch Zugabe von 500 ul in jede Vertiefung. Geschirr Inkubation 30 min bei RT. </li><li> Saugen Sie den Poly-L-Lysin-Lösung und spülen Sie die Platte mit sterilem Wasser. </li><li> Nächstes Beschichtung der Vertiefungen der Platte mit 500 ul 5 ug / ml Maus-Laminin und Inkubation für 30 min. Nach der Inkubation, Waschen der Vertiefungen mit PBS. </li><li> 4 Tage nach splitting der Zellen (Schritt 1.4), übertragen Sie die kleinen Neurosphären in den Kolben mit der Bezeichnung &quot;Für Differenzierung&quot; in die beschichteten Schalen in einer Dichte von ~ 500 Neurosphären pro Vertiefung einer 24-Well-Platte. </li><li> Nach 6 Stunden, wenn die Neurosphären in die Schale legen, entfernen Sie das Proliferationsmedium und fügen Differenzierungsmedium, bestehend aus Neurobasalmedium, B27 ergänzen, NGF (20 ng / ml), BDNF (10 ng / ml), DBC (100 mM), und Retinsäure (2 uM). </li></ol> 3. Immunfluoreszenz-Färbung von Differentiated NPCs (Abbildung 3) <ol><li> Nach Kultur der Zellen in neuroprogenitor neuronalen Differenzierungsmedium für zwei Wochen, wird ein Neuron-reiche Kultur erhalten. Spülen Sie die Neuronen einmal in PBS und dann fix sie in 4% Paraformaldehyd für 30 min. Einmal befestigt, spülen Sie die Zellen dreimal mit PBS. </li><li> Inkubieren der Zellen mit Permeabilisierung Puffer (5% BSA und 0,2% Triton X-100 in PBS) bei Raumtemperatur für 60 min. </li><li> Geschirr O / N Inkubieren bei 4 ° C ina Shaker mit der folgenden Kombination von polyklonalen und monoklonalen Antikörpern in 3% BSA in PBS: NeuN (1:250) und Synapsin (1:250); Acetylcholinesterase (1:500) und Synaptophysin (1:250), BDNF (1 : 500) und GAP43 (1:250); STAT3 (1:500) und GFAP (1:1000). </li><li> Dreimal Waschen Sie die Deckgläser mit PBS inkubiert und dann sie mit Anti-Kaninchen-Cy3-und Anti-Maus-FITC Sekundärantikörper bei RT in Dunkelheit für 90 min. Nach der Inkubation, waschen Sie die Deckgläser dreimal mit PBS. </li><li> Zeigen 10 ul Montagemedium auf einen Objektträger, nehmen Sie das Deckglas von der Kulturschale mit einer Pinzette, und legen Sie es auf den Kopf an der Montage Medium. Sanft, wischen Sie überschüssiges Medium Montage und die Ränder mit Nagellack. </li><li> Untersuchen der immun Neuronen in einem Fluoreszenzmikroskop. </li></ol> 4. Laser Mikrodissektion von Neuronen (Fig. 4) <ol><li> Differenzieren NPCs in einem Neuron-reiche Kultur auf PEN-Membran SchieberS mit Poly-L-Lysin und Laminin der Maus nach den für Fig. 2 beschriebenen Verfahren beschichtet. </li><li> Führen Sie alle weiteren Schritte unter RNase-freien Bedingungen. </li><li> Stain die Kulturen mit HistoGene Stain (Arcturus), um die Zellen zu visualisieren. </li><li> Finden Sie die Bereiche der reinen neuronalen Populationen frei von Astrozyten mit der Straßenkarte Bild des gesamten Folie mit dem Veritas LCM-System. Markieren Sie die Neuronen mit Hilfe der Zeichenwerkzeuge. </li><li> Laser-Capture durchführen dieser Bereiche mit computergesteuerten Präzisions und Automatisierung in einem zweistufigen Prozess. Zuerst wird IR (Infrarot) an mehreren Stellen mit einer Laserleistungseinstellung von 70 mW und einer Puls 2.500 &amp;mgr; gebrannt, um die Membran zu befestigen, um die Kappe. Als nächstes wird der markierte Bereich unter Verwendung eines UV-Schneidwerkzeug bei einem niedrigen Pegeleinstellung von 10 mV ausgeschnitten. </li><li> Kombination dieser Verfahren selektiv erfasst die markierten Bereiche aus der PEN-Membran auf CapSure LCM Makro Kappen. Wenn die Kappe angehoben wird, die Membran mit dem neurons hält sich an die Mütze. </li><li> Isolieren Gesamt-RNA aus LCM-Proben unter Verwendung eines PicoPure RNA-Isolierung-Kit (Arcturus) und Behandlung mit DNase. </li><li> Amplify die isolierte RNA mit RNA-Amplifikation Kit RiboAMP folgenden Anweisungen aus dem Kit. </li><li> Führen Sie Echtzeit-RT-PCR-Analyse unter Verwendung von TaqMan-Sonden für die menschliche Neurofilament schweren Kette (hNFHc) zu erfassen. </li></ol> Abkürzungen: AD, Alzheimer-Krankheit, BDNF, brain-derived neurotrophic factor, CREB, cyclische AMP-Antwortelement bindendes Protein, DBC, cyclische Dibutyryl AMP, EGF, epidermaler Wachstumsfaktor, FGF, Fibroblastenwachstumsfaktor, GFAP, saures Gliafaserprotein; LCM Laser Mikrodissektion, NGF, Nervenwachstumsfaktor; NPC, neuroprogenitor Zelle; PEN, Polyethylennaphthalat.

<ol>
	<li>Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+identity+of+neural+stem+cells.">The glial identity of neural stem cells.</a> Nat Neurosci. 6, 1127-1134 (2003).</li><li>Galli, R., Gritti, A., Bonfanti, L., Vescovi, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells:+an+overview.">Neural stem cells: an overview.</a> Circ. Res. 92, 598-608 (2003).</li><li>Kim, S. U., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+cell+therapy+in+neurological+diseases:+a+review.">Stem cell-based cell therapy in neurological diseases: a review.</a> J. Neurosci. Res. 87, 2183-2200 (2009).</li><li>Breier, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cells+as+models+for+high-throughput+screens+of+developmental+neurotoxicity:+state+of+the+science.">Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: state of the science.</a> Neurotoxicol. Teratol. 32, 4-15 (2010).</li><li>Christie, V. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid+supplementation+of+differentiating+human+neural+progenitors+and+embryonic+stem+cells+leads+to+enhanced+neurogenesis+in+vitro.">Retinoid supplementation of differentiating human neural progenitors and embryonic stem cells leads to enhanced neurogenesis in vitro.</a> J Neurosci Methods. 193, 239-245 (2010).</li><li>Bonner, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Vincent, V. A., DeVoss, J. J., Ryan, H. S., Murphy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neuronal+gene+expression+with+laser+capture+microdissection.">Analysis of neuronal gene expression with laser capture microdissection.</a> J. Neurosci. Res. 69, 578-586 (2002).</li><li>Robles, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+gene+expression+profiling+in+spatial+discrimination+learning.">Hippocampal gene expression profiling in spatial discrimination learning.</a> Neurobiol. Learn Mem. 80, 80-95 (2003).</li><li>Su, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+ethanol+versus+formalin+fixation+on+preservation+of+histology+and+RNA+in+laser+capture+microdissected+brain+tissues.">Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.</a> Brain Pathol. 14, 175-182 (2004).</li><li>Shimamura, M., Garcia, J. M., Prough, D. S., Hellmich, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+and+analysis+of+amplified+antisense+RNA+from+distinct+cell+populations+of+the+young+and+aged+rat+brain:+effect+of+traumatic+brain+injury+on+hippocampal+gene+expression.">Laser capture microdissection and analysis of amplified antisense RNA from distinct cell populations of the young and aged rat brain: effect of traumatic brain injury on hippocampal gene expression.</a> Brain Res. Mol. Brain Res. 122, 47-61 (2004).</li><li>Pugazhenthi, S., Wang, M., Pham, S., Sze, C. I., Eckman, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+CREB+expression+in+Alzheimer's+brain+and+in+Abeta-treated+rat+hippocampal+neurons.">Downregulation of CREB expression in Alzheimer's brain and in Abeta-treated rat hippocampal neurons.</a> Mol. Neurodegener. 6, 60 (2011).</li><li>Dworkin, S., Mantamadiotis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+CREB+signalling+in+neurogenesis.">Targeting CREB signalling in neurogenesis.</a> Expert Opin. Ther. Targets. 14, 869-879 (2010).</li><li>Trujillo, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+perspectives+of+neural+stem+cell+differentiation:+from+neurotransmitters+to+therapeutics.">Novel perspectives of neural stem cell differentiation: from neurotransmitters to therapeutics.</a> Cytometry A. 75, 38-53 (2009).</li><li>Pugazhenthi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Varicella-zoster+virus+infection+of+differentiated+human+neural+stem+cells.">Varicella-zoster virus infection of differentiated human neural stem cells.</a> J. Virol. 85, 6678-6686 (2011).</li><li>Kamme, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microarray+analysis+in+hippocampus+CA1:+demonstration+and+validation+of+cellular+heterogeneity.">Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity.</a> J. Neurosci. 23, 3607-3615 (2003).</li></ol>

Interessenkonflikte zu erklären.

Wir beschreiben in dieser Studie ein Neuron-reichen Zellkulturmodell durch die Differenzierung von selbst erneuernden menschlichen neuroprogenitor Zellen und eine Methode, um eine reine Population von Neuronen durch Laser-Mikrodissektion isolieren. Wir haben eine Kombination von NGF, BDNF, DBC und Retinsäure für die neuronale Differenzierung von NPCs verwendet. DBC wird zur CREB, ein Transkriptionsfaktor, der Neurogenese 12 verbessert aktivieren. Retinsäure induziert Zellzyklusaustritt und reduziert die Gl...

Lebenslange Neurogenese ist bekannt, in der Subventrikularzone der Seitenventrikel und der subgranulären Schicht des Gyrus dentatus des erwachsenen Gehirns von Säugetieren 1 auftreten. Neuroprogenitor Zellen (NPCs), die aus diesen Regionen stammen, sind multipotente Zellen, die in Neuronen, Astrozyten und Oligodendrozyten 2 differenzieren können. NSC haben Interesse, weil ihr Potential bei Patienten mit verschiedenen neurodegenerativen Erkrankungen einschließlich Parkinson-Krankheit, amyotrophe Lateralsklerose, Schlaganfall und Alzheimer-Krankheit (AD) 3 transplantiert werden erzeugt. Studien mit NPCs haben in der Regel auf dieser Transplantation Winkel konzentriert, aber das Potential der NPC-abgeleiteten Neuronen als Zellkultur-Modell, um den Mechanismus der Neurodegeneration zu bestimmen, ist noch nicht vollständig ausgenutzt. Frühere Studien haben in der Regel verwendet, post-mitotischen Neuronen aus Nager-Hirngewebe, die für jedes Experiment isoliert werden müssen, da sie nicht isoliert sindSelbsterneuerung. Obwohl humane Neuroblastom-Zelllinien, einschließlich SH-SY5Y-und SK-N-MC-Zellen erweitert werden kann, haben sie nicht die Eigenschaften der primären Neuronen. Menschliche NSC, andererseits bieten sowohl Vorteile, da sie für mehrfache Durchgänge erweitert werden und differenziert werden, um eine Zellpopulation mit den Merkmalen des primären Neuronen 4,5 zu erzeugen. In der aktuellen Studie beschreiben wir eine neue Differenzierungsprotokoll, um eine konsistente Neuron-reichen Bevölkerung aus kommerziell erhältlichen NPCs aus menschlichen fetalen Gehirns isoliert zu erhalten. Da diese Kulturen haben einen kleinen Prozentsatz von Gliazellen enthalten, brauchen wir zusätzliche Methoden, um eine reine Population von Neuronen zur molekularen Charakterisierung isolieren. Laser-Capture-Mikrodissektion (LCM) ist eine neuartige Technik, mit der eine homogene Population von Zellen aus einem Gewebeschnitt selektiv zur Genexpressionsanalyse 6 erfasst werden. Das Gehirn ist ein heterogenes Gewebe, bestehend aus Neuronen, Glia und anderen Zell types. LCM wurde verwendet, um Neuronen-spezifische Genexpression zu bestimmen, analysiert 7-10. Wir haben schon früher durchgeführt LCM von Neuronen im Hippocampus von AD (Tg2576) Maushirnschnitten zu einer verminderten Expression des zyklischen AMP-Response-Element-bindendes Protein (CREB) und BDNF spezifisch in Neuronen im Hippocampus 11 zeigen. In der vorliegenden Studie beschreiben wir Verfahren für die Erweiterung der menschlichen NPCs, neuronale Differenzierung, Immunfluoreszenzfärbung für neuronale Marker und LCM für die Isolierung von Neuronen auf PEN-Membran-Folien kultiviert.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Neuroprogenitor cells (NPCs) </td>
			<td>Lonza</td>
			<td>PT-2599</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A human basal medium</td>
			<td>Stem cell technology</td>
			<td>5750</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Proliferation supplement</td>
			<td>Stem cell technology</td>
			<td>5753</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Differentiation supplement</td>
			<td>Stem cell technology</td>
			<td>5754</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B-27 Supplement (50x) </td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human epidermal growth factor</td>
			<td>Stem cell technology</td>
			<td>2633</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fibroblast growth factor </td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Brain Derived Neurotrophic Factor </td>
			<td>Cell signaling </td>
			<td>3897S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nerve Growth Factor </td>
			<td>Invitrogen</td>
			<td>13257-019</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibutyryl cyclic AMP</td>
			<td>Sigma</td>
			<td>D-0627</td>
			<td></td>
		 </tr>
		 <tr>
			<td>poly-L-lysine </td>
			<td>Sigma</td>
			<td>P-5899</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse laminin </td>
			<td>Sigma</td>
			<td>L-2020</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R-2625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>STAT3 antibody</td>
			<td>Cell signaling</td>
			<td>9132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFAP antibody</td>
			<td>Cell signaling</td>
			<td>3670</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GAP43 antibody</td>
			<td>Transduction laboratories</td>
			<td>612262</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BDNF antibody</td>
			<td>Millipore</td>
			<td>AB1534SP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetyl cholinesterase antibody </td>
			<td>Santz cruz</td>
			<td>Sc-11409</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synaptophysin antibody</td>
			<td>Abcam</td>
			<td>ab18008-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NeuN antibody</td>
			<td>Chemicon</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synapsin antibody</td>
			<td>Novus</td>
			<td>NB300-104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse FITC</td>
			<td>Jackson Immuno </td>
			<td>115-095-146</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti Rabbit Cy3</td>
			<td>Jackson Immuno </td>
			<td>711-165-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triton-X 100</td>
			<td>Acros</td>
			<td>21568-2500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Paraformaldehye </td>
			<td>Fisher</td>
			<td>4042</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip (Big circle cover slip)</td>
			<td>Fisherbrand</td>
			<td>12-545-102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mounting medium (Prolong Gold)</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen membrane</td>
			<td>Applied biosystems</td>
			<td>LCM0521</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Histogene LCM frozen section staining kit</td>
			<td>Applied biosystems</td>
			<td>KIT0401</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboAmp RNA Amplification kit</td>
			<td>Applied biosystems</td>
			<td>KIT0201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Picopure RNA Isolation kit</td>
			<td>Applied biosystems</td>
			<td>KIT0202</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CapsureMacro LCM caps</td>
			<td>Applied biosystems</td>
			<td>LCM0211</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture

Neuroprogenitor Zellen (NPC) von der menschlichen fötalen Gehirn isoliert wurden unter proliferativen Bedingungen in Gegenwart von epidermalem Wachstumsfaktor (EGF) und Fibroblastenwachstumsfaktor (FGF), um eine reichliche Zufuhr von Zellen bereitzustellen erweitert. NSC in Gegenwart einer neuen Kombination von Nervenwachstumsfaktor (NGF) differenziert, brain-derived neurotrophic factor (BDNF), Dibutyryl-cAMP (DBC) und Retinsäure auf mit Poly-L-Lysin-und Maus-Laminin beschichteten Platten zu erhalten Neuron reichen Kulturen. NPCs wurden auch in der Abwesenheit von Neurotrophine, DBC und Retinsäure und in Gegenwart von ciliary neurotrophic factor (CNTF) zu Astrozyten-reichen Kulturen ergeben differenziert. Differenzierte NPCs wurden durch Immunfluoreszenzfärbung für ein Panel von neuronalen Marker NeuN einschließlich, Synapsin, Acetylcholinesterase, Synaptophysin und GAP43 gekennzeichnet. Saure Gliafaserprotein (GFAP) und STAT3, Astrozyten-Marker wurden in 10-15% der differenzierten NPCs erkannt. Zur ErleichterungZelltyp-spezifische molekulare Charakterisierung wurde die Laser Mikrodissektion durchgeführt, um Neuronen auf Polyethylennaphthalat (PEN)-Membran gleitet kultiviert isolieren. Die in dieser Studie beschriebenen Methoden liefern wertvolle Werkzeuge, um unser Verständnis der molekularen Mechanismen der Neurodegeneration zu fördern.

Ausbau der NPCs (Abbildung 1) Wenn Neurosphären werden auf einer Einzelzellsuspension durch Verreiben gebrochen, muss der Pipettenspitze, um den Boden des Röhrchens zu berühren, so dass es einen gewissen Widerstand, wenn die Suspension auf und ab pipettiert. Die Anzahl der Verreibung wird zwischen Individuen und muß durch Versuch und Irrtum durch Untersuchen der resultierenden Zellsuspension unter dem Mikroskop bestimmt werden variieren. Es ist kritisch, um eine ausreichend...

Watch this Scientific Journal Video about Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture at JoVE.com

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Menschen neuroprogenitor Zellen (NPCs) wurden unter proliferierenden Bedingungen erweitert. NSC wurden in Neuron-reiche Kulturen in der Gegenwart einer Kombination von Neurotrophinen unterscheiden. Neuronale Marker wurden durch Immunfluoreszenz-Färbung nachgewiesen. Um eine reine Population von Nervenzellen zu isolieren, wurden NPCs auf PEN-Membran-Folien differenziert und Laser-Mikrodissektion durchgeführt wurde.

Laser Capture Mikrodissektion von Neuronen aus Differenzierte Menschen Neuroprogenitor Zellen in Kultur

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

laser-capture-microdissection-neurons-from-differentiated-human

Research

JoVE Journal

Neuroscience

12.1K Aufrufe.  Denver VA Medical Center.  Menschen neuroprogenitor Zellen (NPCs) wurden unter proliferierenden Bedingungen erweitert. NSC wurden in Neuron-reiche Kulturen in der Gegenwart einer Kombination von Neurotrophinen unterscheiden. Neuronale Marker wurden durch Immunfluoreszenz-Färbung nachgewiesen. Um eine reine Population von Nervenzellen zu isolieren, wurden NPCs auf PEN-Membran-Folien differenziert und Laser-Mikrodissektion durchgeführt wurde.

Serie: Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

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

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Laser Capture Mikrodissektion von Drosophila Peripheren Neuronen

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Effiziente Ableitung von humanen neuronalen Vorläuferzellen und Neuronen aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Laser Capture Mikrodissektion von Angereichert Populationen von Neuronen oder Single Neurons zur Genexpressionsanalyse Nach Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Produktion von RNA für die Transkriptomische Analyse von Maus Spinal Cord Motor Neuron Cell Bodies durch Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Riechzellen durch nasale Biopsie Combined Erhalten mit Laser-Mikrodissektion: Ein möglicher Ansatz zum Behandlungserfolg bei Mental Disorders Studieren

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser Mikrodissektion - Eine Demonstration der Isolation der einzelnen Dopamin-Neuronen und die gesamte ventrale Tegmentum

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsalwurzelganglien Neuronen und differenzierten Fettgewebe gewonnene Stammzellen: Eine<em&gt; In-vitro-</em&gt; Co-Kultur Modell zur Regeneration peripherer Nerven Studieren

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Stereotaktische Atlas-geführte Laser Capture Microdissection Hirnregionen von traumatischen Verletzungen betroffen

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

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 ...