Name: laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture
Uploaded: 2013-09-16T14:30:00
Description: Watch this Scientific Journal Video about Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture at JoVE.com

Dette arbejde blev støttet af Merit anmeldelse tilskud (NEUD-004-07F) fra Veterans Administration (til SP).

1.. Udvidelse af menneskelige NPCs (figur 1) <ol><li> Genoplive den frosne bestand af menneskelige NPCs fra fostrets hjerne (Lonza, Walkersville, MD, USA) og kultur dem i suspension i T-75 kolber som neurosfærer (figur 1a) i neurobasalt medium indeholdende spredning kosttilskud, EGF (10 ng / ml) og FGF (10 ng / ml). </li><li> Efter 3 dage i kultur overføre neurosfærerne til et 15 ml rør og centrifugeres ved 500 rpm i 5 min. </li><li> Supernatanten efterlader ~ 100 ul medium over c...

<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. 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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. 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Vi beskriver i denne undersøgelse en neuron-rige cellekulturmodel ved differentiering af selvfornyende humane neuroprogenitor celler og en fremgangsmåde til at isolere en ren population af neuroner ved hjælp af laser capture microdissection. Vi har anvendt en kombination af NGF, BDNF, DBC og retinsyre for neuronal differentiering af NPC. DBC anvendes til at aktivere CREB, en transkriptionsfaktor, der øger neurogenese 12. Retinsyre inducerer cellecyklus exit og reducerer glial befolkning 13. Det...

Livslang neurogenese vides at forekomme i subventricular zone af de laterale ventrikler og i subgranular lag af gyrus dentatus fra pattedyrhjernen voksen 1. Neuroprogenitor celler (NPCs), som stammer fra disse regioner er multipotente celler, der kan differentiere sig til neuroner, astrocytter og oligodendrocytter 2. NPC har vakt interesse på grund af deres potentiale til at blive transplanteret i patienter med forskellige neurodegenerative lidelser, herunder Parkinsons sygdom, amyotrofisk lateral sklerose, slagtilfælde og Alzheimers sygdom (AD) 3. Undersøgelser med NPC har generelt været fokuseret på denne transplantation vinkel men potentialet af NPC afledt neuroner som en cellekultur modellen til at bestemme mekanismen af ​​neurodegenerering er ikke blevet fuldt udnyttet. Tidligere undersøgelser har generelt anvendt post-mitotiske neuroner isoleret fra gnaver hjernevæv, der skal isoleres for hvert forsøg, da de ikke erselvfornyende. Selvom menneskelig neuroblastomcellelinier herunder SH-SY5Y og SK-N-MC-celler kan udvides, de ikke har karakter af primære neuroner. Menneskelige NPC&#39;ere, på den anden side, giver både fordele, fordi de kan udvides til flere passager og kan differentieres til at generere en celle population med kendetegnene for primære neuroner 4,5. I den aktuelle undersøgelse, beskriver vi en ny differentiering protokol til opnåelse af en konsekvent neuron-rig bestand fra kommercielt tilgængelige NPC isoleret fra human føtal hjerne. Fordi disse kulturer indeholder en lille procent af gliaceller, vi har brug for yderligere metoder til at isolere en ren population af neuroner til molekylær karakterisering. Laser capture mikrodissektion (LCM) er en hidtil ukendt teknik, som en homogen population af celler fra et vævssnit kan selektivt indfanges til genekspression analyse 6. Hjernen er en heterogen væv bestående af neuroner, glia og anden celle types. LCM er blevet anvendt til at bestemme neuron-specifik genekspression analyser 7-10. Vi har tidligere udført LCM af hippocampale neuroner fra AD (Tg2576) musehjerne sektioner til at vise reduceret ekspression af cyklisk AMP-responselement bindingsprotein (CREB) og BDNF specifikt i hippocampale neuroner 11. I den foreliggende undersøgelse beskriver vi procedurer for udvidelse af menneskelige NPC&#39;ere, neuronal differentiering, immunfluorescensfarvning for neuronale markører og LCM til isolering af neuroner dyrket på PEN membran dias.

<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 celler (NPCs) isoleret fra det menneskelige føtal hjerne blev udvidet under proliferative tilstande i tilstedeværelse af epidermal vækstfaktor (EGF) og fibroblast vækstfaktor (FGF) til at give en rigelig forsyning af celler. NPC blev differentieret i nærvær af en ny kombination af nervevækstfaktor (NGF), hjerne-afledt neurotrofisk faktor (BDNF), dibutyryl cAMP (DBC) og retinsyre på skåle coatet med poly-L-lysin og muselaminin at opnå neuron -rige kulturer. NPC blev også differentieres i fravær af neurotrophiner DBC og retinsyre og i nærvær af ciliær neurotrofisk faktor (CNTF) til opnåelse af astrocyt-rige kulturer. Differentierede NPC&#39;ere var præget af immunfluorescensfarvning for et panel af neuronale markører herunder Neun, synapsin, acetylcholinesterase, synaptophysin og GAP43. Glial fibrillært surt protein (GFAP) og STAT3, astrocyt markører, blev påvist i 10-15% af differentierede NPC. For at lettecelletypespecifik molekylær karakterisering blev laser capture microdissection udført for at isolere neuroner dyrket på polyethylennaphthalat (PEN) membran lysbilleder. De er beskrevet i denne undersøgelse metoder giver værdifulde værktøjer til at fremme vores forståelse af den molekylære mekanisme af neurodegeneration.

Udvidelse af NPC (figur 1) Når neurosfærer er brudt ned til en enkelt cellesuspension ved triturering, pipettespidsen nødt til at røre bunden af ​​røret, så at der er en vis modstand, når suspensionen pipetteres op og ned. Antallet af gange af trituration vil variere mellem individer og skal besluttes ved trial and error ved at undersøge den resulterende celle suspension under et mikroskop. Det er vigtigt at tilvejebringe tilstrækkelig tæthed af cellesuspensionen ...

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

Humane neuroprogenitor celler (NPCs) blev udvidet under prolifererende forhold. NPC blev differentieret i neuron-rige kulturer i nærvær af en kombination af neurotrophiner. Neuronale markører blev påvist ved immunfluorescens-farvning. At isolere en ren population af neuroner blev NPC&#39;ere differentieres på PEN membran dias og laser capture mikrodissektion blev udført.

Laser Capture mikrodissektion af neuroner fra differentierede humane Neuroprogenitor celler i kultur

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

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Produktion af RNA til transskriptionsanalyse fra Mus Rygmarv Motor Neuron Cell Organer ved Laser Capture Microdissection

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

Olfaktoriske Neuroner opnået ved nasal biopsi Kombineret med Laser-Capture mikrodissektion: En Potentiel tilgang til Uddannelse behandlingsrespons i Mental Disorders

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

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Laser Capture mikrodissektion - en demonstration af Isolering af Individual dopaminneuroner og hele Ventral tegmentalområde

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

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

Dorsalrodsganglier neuroner og differentierede adipøst afledte stamceller: An<em&gt; In vitro</em&gt; Co-kultur Model til Uddannelse Perifere nerveregenerering

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 ...