Name: isolation and culture of embryonic mouse neural stem cells
Uploaded: 2018-11-11T14:30:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Embryonic Mouse Neural Stem Cells at JoVE.com

Este trabalho foi financiado pelo Instituto de Royan.

Todas as cirurgias e procedimentos em animais, foram aprovados pelo Comitê de ética animal do Instituto de Royan, Teerã, Irã.
1. preparação dos instrumentos cirúrgicos, lavagem Buffer (HEPES-MEM), meios de cultura celular e placas de cultura de células
<ol><li>Esterilize os instrumentos cirúrgicos (tesouras, punho de lâmina de bisturi e pinça) por autoclave-los baseados nas orientações da rotina de esterilização.</li>
	<li>Preparar um volume adequado de tampão HEPES-MEM (cerc...

<ol>
	<li>Navarro Quiroz, E., 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=Cell+Signaling+in+Neuronal+Stem+Cells.">Cell Signaling in Neuronal Stem Cells.</a> Cells. 7 (7), (2018).</li><li>Choi, C. I., 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=The+Thrombin+Receptor+Restricts+Subventricular+Zone+Neural+Stem+Cell+Expansion+and+Differentiation.">The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation.</a> Scientific Reports. 8 (1), 9360 (2018).</li><li>Li, H., 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=Isolation+of+a+novel+rat+neural+progenitor+clone+that+expresses+Dlx+family+transcription+factors+and+gives+rise+to+functional+GABAergic+neurons+in+culture.">Isolation of a novel rat neural progenitor clone that expresses Dlx family transcription factors and gives rise to functional GABAergic neurons in culture.</a> Developmental Neurobiology. 72 (6), 805-820 (2012).</li><li>Liu, 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=Derivation+of+phenotypically+diverse+neural+culture+from+hESC+by+combining+adherent+and+dissociation+methods.">Derivation of phenotypically diverse neural culture from hESC by combining adherent and dissociation methods.</a> Journal of Neuroscience Methods. , (2018).</li><li>Dhara, S. K., 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=Human+neural+progenitor+cells+derived+from+embryonic+stem+cells+in+feeder-free+cultures.">Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures.</a> Differentiation. 76 (5), 454-464 (2008).</li><li>Aligholi, H., Hassanzadeh, G., Gorji, A., Azari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Biopsy+Method+for+Isolating+Neural+Stem+Cells+from+the+Subventricular+Zone+of+the+Adult+Rat+Brain+for+Autologous+Transplantation+in+CNS+Injuries.">A Novel Biopsy Method for Isolating Neural Stem Cells from the Subventricular Zone of the Adult Rat Brain for Autologous Transplantation in CNS Injuries.</a> Methods in Molecular Biology. 1462, 711-731 (2016).</li><li>Azari, H., Sharififar, S., Rahman, M., Ansari, S., Reynolds, B. 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M., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+multipotent+neural+stem+or+progenitor+cells+from+both+the+dentate+gyrus+and+subventricular+zone+of+a+single+adult+mouse.">Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse.</a> Nature Protocols. 7 (2012), (2005).</li><li>Liu, P., 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=Passage+Number+is+a+Major+Contributor+to+Genomic+Structural+Variations+in+Mouse+iPSCs.">Passage Number is a Major Contributor to Genomic Structural Variations in Mouse iPSCs.</a> Stem Cells and Development. 32 (10), 2657-2667 (2014).</li><li>Diaferia, G. R., 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=Systematic+Chromosomal+Analysis+of+Cultured+Mouse+Neural+Stem+Cell+Lines.">Systematic Chromosomal Analysis of Cultured Mouse Neural Stem Cell Lines.</a> Stem Cells and Development. 20 (8), 1411-1423 (2011).</li><li>Hemmer, K., 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=Induced+Neural+Stem+Cells+Achieve+Long-Term+Survival+and+Functional+Integration+in+the+Adult+Mouse+Brain.">Induced Neural Stem Cells Achieve Long-Term Survival and Functional Integration in the Adult Mouse Brain.</a> Stem Cell Reports. 3 (3), 423-431 (2014).</li><li>Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry+Protocols+for+Surface+and+Intracellular+Antigen+Analyses+of+Neural+Cell+Types.">Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types.</a> Journal of Visualized Experiments. (94), 52241 (2014).</li><li>Zhang, Z., Wang, Z., Rui, W., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnesium+lithospermate+B+promotes+proliferation+and+differentiation+of+neural+stem+cells+in+vitro+and+enhances+neurogenesis+in+vivo.">Magnesium lithospermate B promotes proliferation and differentiation of neural stem cells in vitro and enhances neurogenesis in vivo.</a> Tissue and Cell. 53, 8-14 (2018).</li><li>Zilkha-Falb, R., Gurevich, M., Hanael, E., Achiron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prickle1+as+positive+regulator+of+oligodendrocyte+differentiation.">Prickle1 as positive regulator of oligodendrocyte differentiation.</a> Neuroscience. 364, 107-121 (2017).</li><li>Wang, L., 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=Oligodendrocyte+differentiation+from+human+neural+stem+cells:+A+novel+role+for+c-Src.">Oligodendrocyte differentiation from human neural stem cells: A novel role for c-Src.</a> Neurochemistry International. 120, 21-32 (2018).</li><li>Palanisamy, 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=Oxytocin+alters+cell+fate+selection+of+rat+neural+progenitor+cells+in+vitro.">Oxytocin alters cell fate selection of rat neural progenitor cells in vitro.</a> PLoS ONE. 13 (1), e0191160 (2018).</li><li>Llewellyn-Smith, I. J., Basbaum, A. I., Braz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term,+dynamic+synaptic+reorganization+after+GABAergic+precursor+cell+transplantation+into+adult+mouse+spinal+cord.">Long-term, dynamic synaptic reorganization after GABAergic precursor cell transplantation into adult mouse spinal cord.</a> Journal of Comparative Neurology. 526 (3), 480-495 (2018).</li><li>Precious, S. V., 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=FoxP1+marks+medium+spiny+neurons+from+precursors+to+maturity+and+is+required+for+their+differentiation.">FoxP1 marks medium spiny neurons from precursors to maturity and is required for their differentiation.</a> Experimental Neurology. 282, 9-18 (2016).</li></ol>

Usar uma fonte adequada de células-tronco neurais é muito importante para os neurocientistas. Células-tronco neurais pode ser colhidas de diferentes áreas do cérebro do embrião e podem gerar tipos específicos de neurônios e células gliais. Existem vários métodos para a indução da diferenciação de células-tronco neurais para induzi-los a gerar neurônios maduros e células gliais. Há inúmeros relatos indicando que em condições de cultura específica e regimentos fator trófico, poderia gerar neurônios...

Células-tronco neurais (NSCs) residem em diferentes regiões do cérebro adulto e embrião e eles têm uma tendência a gerar diferentes tipos de neurônios e células gliais1. Zona subventricular no cérebro de mamíferos adultos2 e Eminência ganglionar do cérebro do embrião são regiões ricas de NSC3. No cérebro em desenvolvimento, a Eminência ganglionar oferece a maioria dos interneurônios corticais e particularmente gabaérgica interneurônios3. Também existem métodos menos invasivos para a geração de células-tronco neurais de células-tronco embrionárias (CES) ou uso de células-tronco pluripotentes induzidas (iPSCs) que reduzem a demanda por animal. Apesar do fato de que gerar NSCs de CES ou iPSCs é possível4,5, tem algumas vantagens e desvantagens em comparação com o isolamento de células-tronco neurais do adulto ou embrião cérebro6,7, 8. Os protocolos para induzir a diferenciação de CES e iPSCs em direção a fenótipos neuronais são sempre tempo e custo de consumo e a taxa de sucesso (70-80% Nestin células positivas)5 é mais baixo comparada com isolamento direto de NSCs o cérebro animal ( mais de 99% Nestin células positivas)9. Além disso, as células-tronco perdem sua tendência de estabilidade e diferenciação genética após várias passagens10,11. Embora existam outros novos relatórios sobre a conversão direta de células somáticas em NSCs, estas células são geneticamente e não são facilmente acessíveis em cada laboratório12. Portanto, ainda há uma grande demanda por isolamento de células-tronco neurais do cérebro animal; é possível reduzir a quantidade de uso animal, melhorando as técnicas cirúrgicas. Reduzindo o tempo cirúrgico e melhorar as técnicas, é possível manter as células de danos e obter a maior taxa de NSCs de cada animal.
Aqui, apresentamos uma técnica simplificada e reprodutível para isolamento de células-tronco neurais do cérebro de embrião do rato E13 .

<table border="0" cellpadding="0" cellspacing="0" style="width:942px;" width="941">
	<tbody>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">15 mL tubes</td>
			<td dir="LTR" style="width:145px;">Falcon</td>
			<td dir="LTR" style="width:235px;">352097</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">50 mL tubes</td>
			<td dir="LTR" style="width:145px;">Falcon</td>
			<td dir="LTR">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Adson Forceps, 12 cm, Straight</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR">14226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">B27</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">17504-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">bFGF</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">F0291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">bovine serum albumin (BSA)</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">dish 10cm&#160;</td>
			<td dir="LTR" style="width:145px;">Falcon</td>
			<td dir="LTR" style="width:235px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Dressing Forceps, 12.5 cm, Straight</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR">15908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Dumont Tweezers, 11 cm, Straight</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR" style="width:235px;">500342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">EGF</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">E9644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Ethanol</td>
			<td dir="LTR" style="width:145px;">Merck</td>
			<td dir="LTR" style="width:235px;">100983&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Glutamate</td>
			<td dir="LTR" style="width:145px;">Sigma&#160;</td>
			<td dir="LTR" style="width:235px;">G3291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Glutamax</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">goat anti rabbit FITC conjugated secondary antibody</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">AP307F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">goat serum</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Heparin&#160;&#160;</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">h3149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">HEPES</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">83264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">HEPES</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR">90909C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">insulinsyringe with 25-27 gauge Needle&#160;</td>
			<td dir="LTR" style="width:145px;">SUPA medical</td>
			<td dir="LTR">A1SNL127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">laminin</td>
			<td dir="LTR" style="width:145px;">sigma&#160;&#160;&#160;</td>
			<td dir="LTR" style="width:235px;">L2020</td>
			<td></td>
		</tr>
		<tr height="26">
			<td dir="LTR" height="26" style="height:26px;width:389px;">MEM</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">M2279</td>
			<td></td>
		</tr>
		<tr height="26">
			<td dir="LTR" height="26" style="height:26px;width:389px;">N2 supplement</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">NB medium</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">21103-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Non-essential amino acid (NEAA)</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">PBS without Ca and Mg</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR">20012050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Penicilin/ Streptomycin</td>
			<td dir="LTR" style="width:145px;">Gibco</td>
			<td dir="LTR" style="width:235px;">5140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Poly-L-ornithine</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR" style="width:235px;">&#160;P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">rabbit anti mouse beta tubulin-III antibody</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">T2200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">rabbit anti mouse GFAP antibody</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">G4546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">rabbit anti mouse Nestin antibody</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">N5413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Scalpel Handle #3</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR">500236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Scissors curve</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR" style="width:235px;">14396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">Scissors sharp straight</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td dir="LTR">14192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Soybean trypsin inhibitor</td>
			<td dir="LTR" style="width:145px;">Roche</td>
			<td dir="LTR" style="width:235px;">10109886001&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Tissue culture flasks, T25</td>
			<td dir="LTR" style="width:145px;">BD</td>
			<td dir="LTR" style="width:235px;">353014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Tissue culture flasks, T75</td>
			<td dir="LTR" style="width:145px;">BD</td>
			<td dir="LTR" style="width:235px;">353024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Tween 20</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:235px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">Vannas Scissors,&#160; 8 cm, Straight&#160;</td>
			<td dir="LTR" style="width:145px;">WPI</td>
			<td>14003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:389px;">&#946;-mercaptoethanol</td>
			<td dir="LTR" style="width:145px;">Sigma</td>
			<td dir="LTR">M6250</td>
			<td></td>
		</tr>
	</tbody>
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isolation and culture of embryonic mouse neural stem cells

Células-tronco neurais (NSCs) são multipotentes e pode dar origem a três tipos principais de célula do sistema nervoso central (SNC). Cultura in vitro e expansão de NSCs fornecem uma fonte adequada de células para neurocientistas estudar a função dos neurônios e células gliais, juntamente com suas interações. Existem várias técnicas relatadas para o isolamento de células-tronco neurais do cérebro de mamíferos adultos ou embrião. Durante a operação microcirúrgica para isolar NSCs de diferentes regiões do SNC embrionário, é muito importante reduzir os danos ao cérebro células para obter o rácio mais elevado de células-tronco ao vivo e expansíveis. Uma possível técnica para redução de estresse durante o isolamento dessas células do cérebro do rato embrião é a redução do tempo cirúrgico. Aqui, vamos demonstrar uma técnica desenvolvida para rápida isolamento dessas células da Eminência ganglionar embrião de rato E13 . Os procedimentos cirúrgicos incluem a colheita de embriões de rato E13 do útero, cortando a fontanela frontal do embrião com uma ponta de agulha torta, extraindo o cérebro do crânio, microdissection do cérebro isolado para colher a Eminência ganglionar, dissociação do tecido colhido no meio de NSC para obter uma suspensão de célula única e, finalmente, chapeamento de células em cultura de suspensão para gerar neurospheres.

Micro-dissecção, cela de isolamento e cultura de Neurosphere. Aqui, apresentamos um método rápido e eficiente para o isolamento de células-tronco neurais e Microcirurgia de cérebro do rato E13 . Este artigo mostra que é possível remover o cérebro de um crânio de embrião de rato de13 E através de uma ruptura bem na fontanela frontal. Com esse método, o cérebro sofre menos dano e é possível isolar as células de diferentes partes do cé...

Watch this Scientific Journal Video about Isolation and Culture of Embryonic Mouse Neural Stem Cells at JoVE.com

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Aqui, apresentamos uma nova técnica microcirúrgica para o isolamento de células-tronco neurais da Eminência ganglionar embrião de rato E13 .

Isolamento e cultura de células-tronco neurais embrionárias de rato

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

O objetivo deste protocolo de vídeo é estabelecer um protocolo de origem e eficiente para o isolamento e cultura das células-tronco neurais do rato embrionário. Olá, sou Maryam Sadeghi-Zadeh. Sou um estudante de mestrado em Biologia Celular e Molecular no Instituto Royan.Hoje eu e meu colega queremos mostrar como colher células-tronco neurais de 13 eminência gangliônica de camundongos embriões. Olá, eu sou Bahareh Alizadeh-Shoorjestan. Sou um estudante de mestrado em Biologia Celular e Molecular no Departamento de Células-Tronco do Instituto Royan de Biotecnologia.Olá, eu sou Reza Dehghani-Varnamkhasti. Também estou estudando Biologia Celular e Molecular no Instituto Royan. Os procedimentos cirúrgicos incluem a colheita de cada 13 embriões de camundongos a partir do útero.Cortando a frente do fontanel do embrião com uma ponta de agulha dobrada extraindo o cérebro do crânio. Micro-dissecção do cérebro isolado para colher a eminência gangliônica. Dissociação do tecido colhido em um meio de células-tronco neurais para ganhar uma única suspensão celular.E, finalmente, células de revestimento em uma cultura de suspensão para gerar células nervosas. Limpe e desinfeta a pele da área do abdômen pulverizando com 70% de etanol. Controle a pele para cima do abdômen e, em seguida, corte a pele e sublinhado facial com tesoura para descobrir cavidade abdominal e chifres uterinos.Remova os chifres uterinos contendo os embriões por tesoura e transfira-os para um tubo cônico de 50 ml contendo 25 ml de tampão HEPES MEM frio. Coloque o tubo cônico sob a coifa de fluxo laminal. Abra a tampa debaixo do capô.Retire os chifres uterinos do tubo e enxágue três vezes com volume suficiente do tampão HEPES MEM fresco para eliminar detritos e sangue. Transfira o tecido uterino para uma placa de petri de 10 cm contendo tampão HEPES MEM frio de 7 a 10 ml. Abra os chifres uterinos usando uma tesoura fina e transfira embriões para um novo prato contendo 7 a 10 ml de tampão HEPES MEM frio.Agora coloque o prato de 10 cm com embriões sob o microscópio dissecando para operação de micro-dissecção. Dobre a ponta de uma agulha de seringa empurrando sua ponta em uma alça de lâmina de bisturi de aço. Dobre a ponta da agulha até que a ponta esteja dobrada em um ângulo de 70 a 90 graus.Verifique a ponta da agulha sob o microscópio de dissecação para ver a curva na ponta da agulha. Naturalmente embriões têm uma posição lateral nisso. Sem mudar de posição, segurou o pescoço e o corpo do embrião com fórceps finos e, em seguida, inserir a parte dobrada da agulha profundamente na frente do fontanel da cabeça do embrião.Haveria um pequeno sangramento ou um coágulo de sangue lá. Mova a ponta da agulha superficialmente enquanto a parte torta está dentro da protuberância em direção à testa e, em seguida, em direção à parte de trás da cabeça. Certifique-se de cortar a pele e o crânio, e não danificar o cérebro subjacente.Empurre a pele com a ponta da agulha lateralmente para descobrir o cérebro. Insira a agulha do lado lateral da pele para o lado inferior da estrutura do lado lateral da pele à área abaixo do cérebro. E então remova o cérebro deste couro cabeludo empurrando-o para sair do crânio dissecado.A eminência gangliônica é claramente mostrada em vídeo com suas partes medial e lateral. Manteve o cérebro estável usando as fórceps de cuidado e, em seguida, usando microscissores para cortar o córtex de cada hemisfério para expor a eminência gangliônica. Segurou o cérebro e o lugar dissecado eminência gangliônica no tubo de centrífugas de 15 ml contendo mídia neural de células-tronco.Repita este procedimento até que todo o cérebro tenha sido micro-dissecado. Corte dissecando eminência gangliônica colocando a ponta de pipeta contra a parte inferior do tubo e pipeta a suspensão para cima e para baixo por 25 vezes para quebrar o tecido para obter uma única suspensão celular. Centrifugar a suspensão a 110 g por 5 minutos.Remova o supernasce e resuspense as células em 1 ml de tubo de 0,05% em EDTA. Incubar a suspensão celular em 37 graus Celsius por 10 minutos. E, em seguida, adicionar um volume equivalente de inibidor de trippsina de soja para parar a atividade de trippsina.Pipeta a suspensão da célula para cima e para baixo para ter certeza de que a trippsina foi totalmente inativada. Centrifugar a suspensão da célula a 110 g por 5 minutos. Remova o supernasce e resuspense as células em 1 ml de meio completo de células-tronco neurais e conte o número de células.Plaque as células no meio de células-tronco neurais no frasco de cultura tecidual de tamanho adequado. Transfira 5 ml de 2T25, 20 ml para T75 e 40 ml para frascos T175. Neuroesferas aparecerão após 3 dias de cultura a 37 graus Celsius em uma incubadora umidificada com 5%de CO2.Para sugar a cultura das neurosferas, transfira o meio com neuroesferas suspensas para a centrífuga para centrífuga a 110 g por 5 minutos, e depois descarte o sobrenante. A 1 ml de 0,05% trypsin EDTA e incubar a 37 graus Celsius por 10 minutos. Em seguida, inativar a trippsina usando inibidor de tyrpsina de soja e pipeta as neuroferas suavemente para cima e para baixo para torná-las células únicas.Centrifugar a suspensão da célula a 110 g por 5 minutos. Descarte o supernasce e resuspenque as células em 1 ml de células-tronco neurais médias. Essas células estão prontas para o próximo passo cultura ou cultura em superfície laminada e totalmente revestida para experimentos avançados.Ao cultivar um milhão de células-tronco neurais primárias após sete dias, o número de células aumentará para três a quatro milhões. Após seis a sete dias, as esferas devem medir entre 150 a 200 micrômetros de diâmetro e estarão prontas para subcultura em pratos revestidos de poli ornitina laminados na ausência de bFGF e EGF para diferenciação neuronal. Os resultados do ensaio de citometria de fluxo mostram que cerca de 95% das células que constituem as neurosferas eram Nestin positivo, que é um marcador conhecido para células-tronco neurais.Ensaios usando anticorpos de proteína beta tubulina III e ganglio fibroso revelam que, cultivando células-tronco neurais na superfície revestida cerca de 94% mostraram fenótipo neuronal e cerca de 5% mostram fenótipo gliano. Apenas neste curto vídeo uma técnica de laboratório para o rápido isolamento de células-tronco neurais de 13 eminência gangliônica de embrião de camundongos. Espero que tenha sucesso em sua cultura de células-tronco neurais.

isolation-and-culture-of-embryonic-mouse-neural-stem-cells

Research

JoVE Journal

Neuroscience

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.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Neurociência

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolamento e Expansão do Rato Adulto células-tronco neurais utilizando o ensaio Neurosphere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Estabelecer embrionárias de rato Cultura de Células-tronco neurais utilizando o ensaio Neurosphere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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.

Isolamento e Cultura de células da crista neural-tronco de folículos capilares humanos

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

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Lectina baseado em isolamento e cultura de motoneurônios embrionárias de rato

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolamento e cultivo de células neurais embrionárias de ratos: um protocolo rápido

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Expansão da embrionárias e adultas células-tronco neurais por<em&gt; In Utero</em&gt; Eletroporação ou injeção estereotáxica Viral

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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.

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Um rato, duas culturas: isolamento e cultivo de células-tronco neurais Adulto de as duas zonas neurogênica Individual Ratos

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...