Este trabalho foi apoiado pela Fundação Nacional de Ciência Natural da China (nº 31501099) e pelo Departamento de Educação da Província de Hubei, China (Nº. Q20191104). E agradecemos ao Professor Wensheng Deng da Universidade de Ciência e Tecnologia de Wuhan por fornecer as linhas de células-tronco embrionárias do rato A2lox.

1. Cultura de células-tronco embrionárias do rato
<ol><li>Prepare 0,1% de pratos ou pratos de cultura celular revestido de gelatina.<ol>
<li>Adicione 2 mL de gelatina esterilizada de 0,1% (0,1% w/v em água) a pratos de cultura celular de 60 mm. Arrase suavemente para garantir até mesmo o revestimento dos pratos da cultura celular.</li>
		<li>Coloque os pratos em uma incubadora de CO2 de 5% a 37 °C e deixe o revestimento por 1h.</li>
		<li>Remova a solução de gelatina de 0,1% antes de semear as células. NOTA: Depois de remover a gelatina, não há necessidade de secar ou lavar os pratos revestidos.</li>
	</ol>
</li>
	<li>Cultura de células-tronco embrionárias do rato (A2lox e 129)<ol>
<li>Incubar células mESCs (A2lox e 129) nas células de cultura celular de 0,1% revestidas de 60 mm em meio de crescimento mESC a 37 °C em uma incubadora de CO2 de 5%, respectivamente. O meio de crescimento mESC consiste em 85% de DMEM/F12, 15% de substituição de soro knock-out (KSR), 0,1 mM β-mercaptoethanol (2ME), 2 mM GlutaMAX, 1% aminoácido não essencial (NEAA), 1% penicilina/estreptomicina (P/S), 1000 fator inibidor de leucemia U/mL (LIF), 10 nM CHIR-99021 (inibidor GSK-3) e 0,33 nM PD0325901 (inibidor de MEK). ATENÇÃO: β-mercaptoetanol é inflamável e tem toxicidade por inalação. Mantenha-se afastado das fontes de incêndio e use uma máscara para evitar a inalação quando usar.</li>
		<li>Mude o médio de crescimento do MESC diariamente para um melhor crescimento de A2lox e 129.</li>
		<li>Quando as células atingirem 80% de confluência, retire o meio e adicione 1 mL de 0,1% de trippsina ao prato. Arrase suavemente por 30 s para garantir a cobertura uniforme de trippsina em todas as células.</li>
		<li>Deixe as células por cerca de 1 minuto para tentar e,em seguida, remova a trippsina usando pipeta de 1 mL.</li>
		<li>Adicione 2 mL de mESC médio de crescimento ao prato, pipeta para cima e para baixo várias vezes para fazer uma única suspensão celular.</li>
		<li>Conte a densidade das células na suspensão o mais precisamente possível usando hemótmetro.</li>
		<li>Divida as células em 7 grupos e induz a diferenciação usando diferentes protocolos mostrados na Tabela 1.</li>
	</ol>
</li>
</ol>2. Diferenciação de mESCs para NPCs (Fase I)
<ol><li>Prepare 0,1% de placas de cultura celular revestida de gelatina ou deslizamentos.<ol>
<li>Antes de usar, prepare 0,1% de gelatina revestida de 6 poços ou tampas como na etapa 1.1.</li>
	</ol></li>
	<li>Diferenciação da Fase I utilizando o protocolo 1 (Tabela 1)<ol>
<li>Semente cerca de 2 x 104 mESCs em 2 mL de diferenciação basal médio I por bem nas placas 0,1% revestidas de gelatina 6-well. Verifique a densidade celular sob um microscópio.</li>
		<li>Incubar as células a 37 °C em uma incubadora de CO2 de 5% por 6h para permitir a fixação.</li>
		<li>Após o apego, retire as células da incubadora e lave as células duas vezes com 2 mL de PBS.</li>
		<li>Adicione 2 mL de diferenciação basal médio I(Tabela 1) a cada poço e coloque as células de volta na incubadora.</li>
		<li>Deixe as células para diferenciação por 8 dias. Substitua o meio de diferenciação basal I a cada 2 dias.</li>
	</ol></li>
	<li>Diferenciação da Fase I utilizando o protocolo 2 (Tabela 1)<ol>
<li>Adicione 1,5 x 106 mESCs em um prato bacteriano não adesivo em 10 mL de diferenciação basal médio I para permitir a formação do corpo embrionário a 37 °C em uma incubadora de CO2 de 5%.</li>
		<li>Após 2 dias, a célula de transferência agrega em tubos de centrífugas de 15 mL e os deixa se estabelecer pela gravidade.</li>
		<li>Remova o supernasal e adicione 10 mL de diferenciação basal fresca do meio I para resuspensar os corpos embrióides. Replantá-los em um novo prato bacteriano não adesivo e permitir diferenciação por mais 2 dias.</li>
		<li>Verifique a formação de corpos embrionários sob o microscópio(Figura 2A).</li>
		<li>Recolher corpos embrionários conforme descrito nas etapas 2.3.2-2.3.3. Semente cerca de 50 corpos embrionários em 2 mL de meio de diferenciação basal I por poço em 0,1% de placas revestidas de gelatina 6-well.</li>
		<li>Prepare 1 mM de estoque de RA totalmente trans (em DMSO) e armazene longe da luz em um congelador de -80 °C após a sub-embalagem. NOTA: A RA é instável, e deve-se prestar atenção para mantê-lo longe da luz e reduzir o contato aéreo durante a preparação do estoque de RA.</li>
		<li>Para indução de RA, adicione 2 μL de estoque de RA em cada poço para fazer uma concentração final de 1 μM.</li>
		<li>Coloque a placa na incubadora de CO2 de 5% a 37 °C e diferencie por mais 4 dias.</li>
		<li>Altere todo o 2 mL de diferenciação basal médio I (com RA de 1 μM) a cada 2 dias.</li>
	</ol></li>
	<li>Diferenciação da Fase I utilizando o protocolo 3 (Tabela 1)<ol>
<li>Plante 1,5 x 106 mESCs em um prato bacteriano não adesivo em 10 mL de diferenciação basal médio I. Deixe por 2 dias para formação corporal embrionária a 37 °C em uma incubadora de CO2 de 5%.</li>
		<li>Verifique a formação de corpos embrionários sob um microscópio(Figura 2B).</li>
		<li>Transfira os agregados celulares em tubos de centrífugas de 15 mL e deixe-os se estabelecer pela gravidade.</li>
		<li>Remova o supernatante cuidadosamente e adicione 10 mL de diferenciação basal fresca do meio I para resuspensá-los.</li>
		<li>Semente cerca de 50 corpos embrionários em 2 mL de meio de diferenciação basal I por poço em 0,1% de placas revestidas de gelatina 6-well.</li>
		<li>Para indução de RA, adicione 2 μL de estoque de RA em cada poço para fazer uma concentração final de 1 μM.</li>
		<li>Coloque a placa na incubadora de CO2 de 5% a 37 °C e diferencie por mais 6 dias. Altere todo o meio de diferenciação basal I (com RA de 1 μM) a cada 2 dias.</li>
	</ol></li>
	<li>Diferenciação da Fase I utilizando o protocolo 4 (Tabela 1)<ol>
<li>Semente cerca de 2 x 104 mESCs dentro de 2 mL de meio de diferenciação basal I por bem nas placas de 0,1% revestidas de gelatina 6-well. Coloque a placa na incubadora de CO2 de 5% a 37 °C para permitir a fixação por 6h.</li>
		<li>Após o apego, lave as células duas vezes com 2 mL de PBS. Adicione 2 mL de diferenciação basal médio I a cada poço e deixe a diferenciação por 4 dias na incubadora de 5% de CO2 a 37 °C.</li>
		<li>Para indução de RA, adicione 2 μL de estoque de RA totalmente trans em cada poço (a concentração de trabalho é de 1 μM) para induzir a diferenciação por mais 4 dias.</li>
		<li>Em todo o processo, substitua todo o meio a cada 2 dias.</li>
	</ol></li>
	<li>Diferenciação da Fase I utilizando o protocolo 5 (Tabela 1)<ol>
<li>Semente cerca de 2 x 104 mESCs dentro de 2 mL de meio de diferenciação basal I por bem nas placas de 0,1% revestidas de gelatina 6-well. Coloque a placa na incubadora de CO2 de 5% a 37 °C para permitir a fixação por 6h.</li>
		<li>Lave as células duas vezes com 2 mL de PBS. Adicione 2 mL de diferenciação basal médio I a cada poço e deixe a diferenciação por 2 dias na incubadora de 5% de CO2 a 37 °C.</li>
		<li>Para a indução de RA subsequente, adicione 2 μL de estoque de RA em cada poço para fazer uma concentração final de 1 μM. Coloque a placa na incubadora de CO2 de 5% a 37 °C para induzir a diferenciação por mais 6 dias.</li>
		<li>Em todo o processo, substitua todo o meio a cada 2 dias.</li>
	</ol></li>
	<li>Diferenciação da Fase I utilizando o protocolo 6 (Tabela 1)<ol>
<li>Plante 1,5 x 106 mESCs em um prato bacteriano não adesivo em 10 mL de N2B27 médio II(Tabela 1) para permitir a formação de corpos embrióides.</li>
		<li>No2º dia, recolher os agregados celulares conforme descrito nas etapas 2.3.2-2.3.3 e resuspensar os corpos embrióides utilizando 10 mL de n2B27 médio II fresco.</li>
		<li>Replante-os em um novo prato bacteriano não adesivo e permita diferenciação por mais 2 dias na incubadora de CO2 de 5% a 37 °C. Verifique a formação de corpos embrionários sob microscópio.</li>
		<li>No4º dia, colete corpos embrionários. Semente cerca de 50 corpos embrionários por poço em placas de 6 poços revestidos de gelatina com 2 mL de N2B27 médio II.</li>
		<li>Adicione 2 μL de estoque de RA totalmente trans em cada poço e induza a diferenciação por mais 4 dias. Substitua todo o meio (N2B27 médio II por 1 μM RA) a cada dois dias.</li>
	</ol></li>
	<li>Diferenciação da Fase I utilizando o protocolo 7 (Tabela 1)<ol>
<li>Semente cerca de 2 x 104 mESCs dentro de 2 mL de meio de diferenciação basal I por bem nas placas de 0,1% revestidas de gelatina 6-well. Coloque a placa na incubadora de CO2 de 5% a 37 °C para permitir a fixação por 6h.</li>
		<li>Lave as células duas vezes com PBS. Em seguida, adicione 2 mL de N2B27 médio II a cada poço e deixe a diferenciação por 8 dias a 37 °C em uma incubadora de CO2 de 5%.</li>
		<li>Troque todo o N2B27 médio II a cada 2 dias.</li>
	</ol></li>
</ol>3. Observação da morfologia celular
<ol><li>Verifique o status de diferenciação dos 7 grupos acima mencionados diariamente sob um microscópio de luz de contraste de fase invertida.</li>
	<li>Selecione aleatoriamente pelo menos 12 campos e tire fotos para registrar as alterações morfológicas de cada grupo em D8.</li>
</ol>4. Mancha de imunofluorescência
<ol><li>Preparação da amostra: Seed mESCs em tampas revestidas de gelatina de 0,1% e permitem diferenciação por 8 dias utilizando os protocolos mencionados na etapa 2.</li>
	<li>Enxágüe: No8º dia, retire as amostras da incubadora e remova o meio de diferenciação por aspiração. Enxágue suavemente as células uma vez com 1 mL de PBS por 5 min.</li>
	<li>Fixação: Adicione 1 mL de 4% de paraformaldeído a cada amostra e fixe as células por 20 minutos à temperatura ambiente (TR).</li>
	<li>Enxágüe: Após a fixação, enxágue suavemente as células com 1 mL de PBS 3 vezes, por 5 minutos cada.</li>
	<li>Permeabilização: Adicione 1 mL de 0,2% TritonX-100 em PBS a cada amostra e deixe por 8 min na RT.</li>
	<li>Enxágüe: Após a permeabilização, enxágue suavemente as células com 1 mL de PBS 3 vezes, por 5 minutos cada.</li>
	<li>Bloqueio: Adicione 1 mL de soro de cabra de 10% em PBS a cada amostra e incubar na RT por 1h para bloquear quaisquer interações não específicas.</li>
	<li>Incubação com anticorpo primário<ol>
<li>Diluir o anticorpo anti-Nestin a uma proporção de 1:100 usando 5% de soro de cabra na PBS.</li>
		<li>Aplique 500 μL de anticorpo diluído em diferentes amostras e incubar durante a noite a 4 °C.</li>
	</ol>
</li>
	<li>Enxágüe: Remova o anticorpo e enxágue as amostras suavemente com 1 mL de PBS 3 vezes por 8 minutos cada.</li>
	<li>Incubação com anticorpos secundários<ol>
<li>Diluir o Alexa Fluor 488-rotulado anti-rato IgG com uma proporção de 1:500 usando 5% de soro de cabra em PBS.</li>
		<li>Aplique 500 μL de anticorpo diluído em diferentes amostras e incubar no escuro por 2 h no RT. NOTA: Após aplicar anticorpos secundários fluorescentes, realize todas as etapas subsequentes no escuro para evitar a saciamento da fluorescência.</li>
	</ol>
</li>
	<li>Enxágüe: Remova o anticorpo secundário e enxágue as amostras suavemente com 1 mL de PBS 3 vezes por 8 minutos cada.</li>
	<li>Coloração nuclear e montagem<ol>
<li>Coloque uma gota de meio de montagem DAPI no microslídeo limpo.</li>
		<li>Retire cuidadosamente das amostras das placas e coloque a amostra em cima do meio de montagem da DAPI com a célula de frente para baixo. Deixe no escuro por 5 minutos no RT.</li>
		<li>Remova o excesso de dopi médio de montagem com papel absorvente.</li>
	</ol>
</li>
	<li>Observação de microscopia de fluorescência<ol>
<li>Coloque os espécimes sob a microscopia de fluorescência e detecte o sinal para DAPI e Alexa Fluor 488 usando filtros adequados.</li>
		<li>Escolha uniforme e aleatoriamente 10-15 campos visuais diferentes para cada amostra e grave as imagens com uma câmera CCD.</li>
	</ol>
</li>
</ol>5. Diferenciação de NPCs para neurônios (Fase II)
<ol><li>Prepare os derivados mESC sob a diferenciação da Fase I utilizando o protocolo 3 (8 dias, Tabela 1) conforme detalhado na etapa 2.4, que tem a maior eficiência de diferenciação (Ver Figura 3). NOTA: Após a diferenciação da fase I, deve ser realizado o controle de qualidade utilizando observação de morfologia celular e ensaio de imunofluorescência mencionado acima, para garantir um NPCs saudável e de alto rendimento.</li>
	<li>Semente cerca de 5 x 105 mESC derivados dentro de 2 mL de diferenciação basal médio I por bem nas placas de 0,1% revestidas de gelatina 6-well. Divida aleatoriamente os derivados do mESC em 3 grupos, como o protocolo fase II 1, o protocolo 2 e o protocolo 3, respectivamente.</li>
	<li>Coloque a placa na incubadora de CO2 de 5% a 37 °C para permitir a fixação por 6h. Lave-os duas vezes com 2 mL de PBS.</li>
	<li>Adicione 2 mL de diferenciação basal médio I, N2B27 médio I e N2B27 médio II(Tabela 2), respectivamente,a cada poço dos grupos acima.</li>
	<li>Coloque as placas na incubadora e deixe diferenciar por mais 10 dias. Troque o meio correspondente a cada 2 dias.</li>
	<li>Verifique o estado de diferenciação e regise as alterações morfológicas mencionadas na etapa 3.</li>
	<li>No dia 18, avalie a geração de neurônios (β-Tubulin III positivo) e determine a eficiência de diferenciação dos 3 protocolos utilizando na etapa 4.</li>
</ol>

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E., Li, J., Baccei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Differentiation+of+Retinoic+Acid-Induced+Human+Pluripotent+Embryonic+Carcinoma+Stem+Cell+Neurons.">Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons.</a> PLoS ONE. 6 (1), 16174 (2011).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Developmental Biology. 275, 124-142 (2004).</li></ol>

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No presente estudo, estabelecemos um método simples e eficaz para diferenciação neuronal dos mESCs, com baixo custo e materiais de fácil obtenção. Neste método, 2 dias de formação corporal embrionária seguida de 6 dias de indução de RA podem efetivamente promover a diferenciação de mESCs em NPCs (Fase I-protocolo 3). Para a diferenciação da fase II, o N2B27 médio II (Fase II-protocolo 3) induz mais efetivamente a diferenciação dos NPCs em neurônios. Para garantir o sucesso, mais atenção deve ser dad...

As células-tronco embrionárias (ESCs) são pluripotentes e podem se diferenciar em células precursoras neurais (NPCs) e, posteriormente, em neurônios sob certas condições1. A neurogênese baseada em ESC fornece a melhor plataforma para imitar neurogênese, servindo assim como uma ferramenta útil para estudos de biologia do desenvolvimento e potencialmente auxiliar na medicina regenerativa2,3. Nas últimas décadas, muitas estratégias foram relatadas para induzir neurogênese embrionária, como o método transgênico4, utilizando pequenas moléculas5, utilizando um microambiente de matriz3D 6, e a técnica de cocultura7. No entanto, a maioria desses protocolos são limitados ou difíceis de operar, portanto, não são adequados para uso na maioria dos laboratórios.
Para encontrar um método fácil de operar e de baixo custo para obter uma diferenciação neural eficiente dos mESCs, uma estratégia de triagem combinatória foi usada aqui. Conforme descrito na Figura 1,todo o processo de neurogênese embrionária foi dividido em 2 fases. A fase I refere-se ao processo de diferenciação dos mESCs em NPCs, e a fase II refere-se à diferenciação subsequente dos NPCs em neurônios. Com base nos princípios de fácil operação, baixo custo, materiais facilmente disponíveis e alta eficiência de diferenciação, foram escolhidos sete protocolos na Fase I e três na Fase II com base no sistema tradicional de cultura monocamadas aderente ou no sistema de formação corporal embrionário8,9. A diferenciação de eficiência dos protocolos em ambas as fases foi avaliada por meio da observação da morfologia celular e do ensaio de imunofluorescência. Através da combinação do protocolo mais eficiente de cada fase, estabelecemos o método otimizado para diferenciação neural dos mESCs.

<table>
	<tbody>
		<tr>
			<td>Anti-Nestin antibody [Rat-401]</td>
			<td>Abcam</td>
			<td>Ab11306</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III antibody produced in rabbit</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-Labeled Goat Anti-Mouse IgG</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>stored at -20 &#176;C, and protect from light</td>
		</tr>
		<tr>
			<td>CHIR-99021 (CT99021)</td>
			<td>Selleck</td>
			<td>S1263</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-Labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0516</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>DME/F-12 1:1 (1x)</td>
			<td>HyClone</td>
			<td>SH30023.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Gibco</td>
			<td>CM0635B</td>
			<td>stored at room temperature</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Immunol Staining Primary Antibody dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor human</td>
			<td>Sigma</td>
			<td>L5283</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI - Aqueous, Fluoroshield</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson</td>
			<td>005-000-121</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nonadhesive bacterial dish</td>
			<td>Corning</td>
			<td>3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin Solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PD0325901(Mirdametinib)</td>
			<td>Selleck</td>
			<td>S1036</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>stored at -80 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Strain 129 Mouse Embryonic Stem Cells</td>
			<td>Cyagen</td>
			<td>MUAES-01001</td>
			<td>Maintained in feeder-free culture system</td>
		</tr>
		<tr>
			<td>Stem-Cellbanker (DMSO free)</td>
			<td>ZENOAQ</td>
			<td>stem cellbanker DMSO free</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1X) Solution</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>6 - well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>NUNC</td>
			<td>339650</td>
			<td></td>
		</tr>
	</tbody>
</table>

neuronal differentiation from mouse embryonic stem cells in vitro

A diferenciação neural das células-tronco embrionárias do camundongo (mESCs) é uma ferramenta potencial para elucidar os principais mecanismos envolvidos na neurogênese e potencialmente ajudar na medicina regenerativa. Aqui, estabelecemos um método eficiente e de baixo custo para diferenciação neuronal dos mESCs invitro, utilizando a estratégia de triagem combinatória. Sob as condições aqui definidas, a formação do corpo embrionário de 2 dias + protocolo de indução de ácido retinóico de 6 dias permite diferenciação rápida e eficiente dos mESCs em células precursoras neurais (NPCs), como visto pela formação de A2lox bem empilhados e neuritas semelhantes a 129 derivativos que são nestin positivos. O estado saudável dos corpos embrionários e o ponto de tempo em que o ácido retinóico (RA) é aplicado, assim como as concentrações de RA, são críticos no processo. Na diferenciação subsequente dos NPCs em neurônios, o N2B27 médio II (suplementado pelo meio neurobásal) poderia suportar melhor a manutenção e maturação a longo prazo das células neuronais. O método apresentado é altamente eficiente, de baixo custo e de fácil operação, podendo ser uma poderosa ferramenta para pesquisa em neurobiologia e biologia do desenvolvimento.

Formação corporal embrionária de 2 dias + a indução de RA de 6 dias funciona melhor na direção da diferenciação de mESCs em NPCs (Fase I). Para determinar o protocolo ideal que melhor promove a diferenciação de mESCs em NPCs (Fase I), 7 protocolos foram testados tanto em A2lox quanto em 129 mESCs(Tabela 1) e o status de diferenciação de cada grupo foi monitorado por microscópio leve. Como mostrado na Figura 3A, a maioria dos derivados A2lox e 129 em formação ...

Watch this Scientific Journal Video about Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro at JoVE.com

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Aqui, estabelecemos um método de baixo custo e fácil de operar que direciona a diferenciação rápida e eficiente das células-tronco embrionárias para os neurônios. Este método é adequado para popularização entre laboratórios e pode ser uma ferramenta útil para a pesquisa neurológica.

Diferenciação neuronal de células-tronco embrionárias do rato in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

neuronal-differentiation-from-mouse-embryonic-stem-cells-in-vitro

Research

JoVE Journal

Neuroscience

8.7K visualizações.  Wuhan Center for Disease Control and Prevention. Aqui, estabelecemos um método de baixo custo e fácil de operar que direciona a diferenciação rápida e eficiente das células-tronco embrionárias para os neurônios. Este método é adequado para popularização entre laboratórios e pode ser uma ferramenta útil para a pesquisa neurológica.

Vídeo: Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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.

Derivação eficiente dos recursos humanos Progenitores Neuronal e Neurônios de pluripotentes células estaminais embrionárias humanas com a indução de moléculas pequenas

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

Neurociência

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotípicas culturas de mesencéfalo ventral Slice Embrionárias: Um Sistema para o Estudo de Desenvolvimento Neuronal dopaminérgicos In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dirigido neurônios dopaminérgicos Diferenciação de Células-Tronco pluripotentes humanas

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Diferenciação de células estaminais embrionárias de rato em Cortical interneurônio precursores

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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 expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

Preparação de fatia de cunha in vitro para imitar conectividade do circuito neuronal vivo

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Modelagem in vitro para doenças neurológicas usando conversão direta de fibroblastos para células progenitoras neuronais e diferenciação em astrócitos

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imagem de cálcio em rato de azeitona inferior

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Células Semelhantes à Microglia Humana: Diferenciação de Células-Tronco Pluripotentes Induzidas e Ensaio de Fagocitose de Células Vivas In Vitro usando Sinaptossomos Humanos

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...