Este trabalho foi apoiado por uma subvenção do NIH (1R35GM133496-01) para Z. Gao. Gostaríamos de agradecer ao Dr. Ryan Hobbs pela ajuda na secção. Agradecemos as instalações principais da Penn State College of Medicine, incluindo as Ciências do Genoma e Bioinformática, a Imagem Avançada de Microscopia de Luz e a Citometria de Fluxo. Agradecemos também ao Dr. Yuka Imamura pela assistência na análise do RNA-seq.

1. cultura mESC
<ol><li>Cubra uma placa tratada com 10 cm de tecido com gelatina de 0,1% e deixe que a gelatina se estafina por pelo menos 15-30 minutos antes de aspirar.</li>
	<li>Sementes γ camundongos embrionários (MEFs) um dia antes de cultivar os mESCs no meio mESC pré-aquecido (médio águia modificado de Dulbecco (DMEM) com 15% de soro bovino fetal (FBS), aminoácidos não essenciais, β-mercaptoetanol, L-glutamina, penicilina/estreptomicina, piruvato de sódio, LIF, PD0325901 (PD) e Chir99021 (CH)).</li>
	<li>Permita que os MEFs irradiados de γ se acomodem e se anexem à superfície da placa antes de estancar as células E14.</li>
	<li>Descongele E14 ESCs em banho-maria de 37 °C e transfira rapidamente as células em um tubo cônico de 15 cm com meio mESC quente. Pelota as células a 200 x g por 3 min e remova o supernasce.</li>
	<li>Resuspengem as células em 10 mL de mESC médio e emplaque as células na placa de cultura contendo os MEFs irradiados γ anteriormente. Incubar a cultura celular em uma incubadora de 37 °C abaixo de 5% de CO2.</li>
	<li>Para a passagem de cultura, aspire e o médio e lave a placa com 1x PBS estéril. Adicione 0,05% de trippsina suficiente para cobrir a superfície da placa e incubar a 37 °C por 3 min.</li>
	<li>Neutralizar a trippsina com mESC médio e pipeta para gerar suspensão unicelular. Centrifugar as células a 200 x g por 3 min e remover o supernante.</li>
	<li>Conte as células com um hemótmetro ou contador de células e sementes cerca de 5,0 x 105 células em uma placa de cultura de 10 cm.</li>
	<li>Resuspenque as células em 10 mL de mESC médio, plaque as células na placa de cultura de tecido revestida de gelatina e incuba culturas como descrito anteriormente. NOTA: Recomenda-se que os mESCs sejam aprovados a cada 2 dias para evitar que as células se diferenciem em suas colônias. O fenol vermelho no médio funciona apenas como um indicador de pH e dependendo da densidade celular, pode ficar amarelado (mais ácido), mais cedo do que 2 dias. Portanto, pode ser necessário mudar o meio todos os dias. Os MEFs irradiados γ acabarão morrendo depois de algumas passagens.</li>
</ol>2. EB, NPC e diferenciação de neurônios
<ol><li>Executar o protocolo de passagem de cultura mencionado anteriormente e contar as células (etapas 1.7-1.10).</li>
	<li>Método de queda de suspensão(dia 0)<ol>
<li>Para uma placa de cultura celular de 10 cm, contar cerca de 2,5 x 104 células onde 5,0 x 10 2 célulasserão suspensas em 20 μL de diferenciação (DMEM com 15% de FBS, aminoácidos não essenciais, β-mercaptoetanol, L-glutamina, penicilina/estreptomicina e pirudo de sódio). Cerca de 50 gotículas de 20 μL contendo as células podem ser banhadas em uma placa de 10 cm.</li>
		<li>Alíquotar o número apropriado de células, e então centrifugar as células a 200 x g por 3 min e remover o supernante.</li>
		<li>Resuspenncie as células no volume adequado do meio de diferenciação para uma densidade celular de 5,0 x 102 células por 20 μL (por exemplo, 2,5 x 104 células em 1 mL de meio de diferenciação).</li>
		<li>Usando uma micropipette ou uma pipeta repetidora, coloque 20 gotículas de μL da suspensão celular na tampa da placa de cultura tecidual. Certifique-se de que as gotículas não estão muito próximas umas das outras para evitar que elas se fundam. NOTA: As gotículas podem ser banhadas em uma placa de fixação tratada com cultura tecidual ou placa de suspensão, pois serão colocadas na tampa e não na placa em si. Para uma abordagem mais viável e estéril, plaque as gotículas em uma placa de cultura tecidual de fixação e transfira-as para uma placa de suspensão como descrito abaixo.</li>
		<li>Encha a placa com 5-10 mL de 1x PBS e coloque cuidadosamente a tampa de volta na placa. Incubar a cultura na incubadora de 37 °C. NOTA: O PBS é adicionado à placa de cultura para evitar que as gotículas sequem.</li>
	</ol></li>
	<li>No dia 2,use uma micropipette para coletar as gotículas da tampa e colocá-las em uma placa de suspensão de cultura celular de 10 cm preenchida com 10 mL de meio de diferenciação. Incubar a cultura em um agitador orbital tremendo em baixa velocidade na incubadora.</li>
	<li>No dia 4, para colher os EBs, coletar as células, centrífugas a 200 x g por 3 min, e remover o sobrenante. NOTA: Os EBs também podem ser lavados com 1x PBS de acordo com os requisitos dos experimentos subsequentes.</li>
	<li>Para continuar o procedimento e induzir a diferenciação do EB em células progenitoras neurais (NPCs), prepare o meio de diferenciação com ácido retinóico de 5 μM (RA).</li>
	<li>Remova o meio antigo pelo menos os EBs a 100 x g por 3 min ou deixe que os EBs se acomodem antes de aspirar o meio antigo. Adicione 10 mL do meio de diferenciação contendo RA de 5 μM à placa de cultura.</li>
	<li>No dia 6,substitua pelo menos metade do meio por meio fresco contendo RA de 5 μM, inclinando a placa e pipetando o meio como descrito acima. NOTA: Recomenda-se que pelo menos metade do meio seja substituída por um meio fresco contendo RA nos dias 5 e 7. Tome nota do indicador vermelho fenol; se ficar amarelado, é melhor substituir todo o meio.</li>
	<li>No dia 8,colhe os NPCs coletando as células, centrifugando a 200 x g por 3 min, e removendo o sobrenante. NOTA: Os NPCs também podem ser lavados com 1x PBS de acordo com as necessidades de experimentos subsequentes. Se necessário, os NPCs podem ser congelados e descongelados novamente para cultura e análise posteriores. Se os NPCs forem cultivados, a accutase também pode ser usada como alternativa à trippsina.</li>
	<li>Para continuar o procedimento e diferenciar os NPCs em neurônios, colete NPCs em um tubo cônico de 15 mL por centrifugação, dissociá-los com trippsina e incuba-los a 37 °C por 3 min. Pipetar os NPCs para garantir que todos os agregados de NPC sejam dissociados e neutralizar o trypsin com o meio.</li>
	<li>Filtre as células com coador de células de nylon de 40 μm e conte as células antes de emplacar-as a uma densidade de 1,5 x 105/cm2 em n2 médio (médio DMEM/F12 + 3 mg/mL de glicose + 3 mg/mL rico em lipídios albumina de soro bovino (LBSA) + 1:100 Suplemento N2 + 10 ng/mL bFGF + 50 U/mL pen/strep + 1 mM L-glutamine) em uma placa tratada de cultura tecidual para experimentos subsequentes de PCR e blot ocidental; ou em uma câmara de cultura de tecidos para experimentos de imunofluorescência.</li>
	<li>No dia 9,substitua o antigo meio por um novo meio N2.</li>
	<li>No dia 10,troque o n2 médio com n2/B27 médio (50% DMEM/F12 e 50% basal neural, 3 mg/mL LBA, 1:200 N2 suplemento, 1:100 B27 suplemento, 50 U/mL caneta/estreptococos, e 1 mM L-glutamina).</li>
	<li>Nos dias 11-12,colhe os neurônios da seguinte forma. Lave as células com 1x PBS, adicione trippsina e incuba a cultura na incubadora de 37 °C por 3 minutos antes de neutralizar a trippsina com média e centrífuga a 200 x g por 3 min.</li>
</ol>3. Caracterização de mESCs e células diferenciadas
<ol><li>Ensaio de fosfatase alcalina (AP) 
	<ol>
<li>Use um kit para avaliar a atividade de fosfates alcalina (ver tabela de materiais).</li>
		<li>Retire o meio da placa de cultura e lave os ESCs com 1x PBS.</li>
		<li>Adicione 1 mL da solução fixa (consiste em formaldeído e metanol) fornecido com o kit à placa e incuba-o à temperatura ambiente por 2-5 min. NOTA: A super incubação na solução de correção pode comprometer a atividade AP.</li>
		<li>Remova a solução de correção, lave os ESCs com 1x PBS e deixe alguma quantidade de PBS na placa. NOTA: Mantenha os ESCs úmidos no PBS para não comprometer a atividade ap.</li>
		<li>Prepare a solução AP misturando as soluções de substrato A, B e C na proporção 1:1:1. Misture primeiro as soluções A e B e incubar a mistura à temperatura ambiente por 2 minutos antes de adicionar a solução C.</li>
		<li>Remova o PBS 1x e adicione a solução AP preparada anteriormente.</li>
		<li>Incubar os ESCs por cerca de 15 minutos no escuro, embrulhando a placa de cultura com papel alumínio ou realizando o passo 3.5 em uma sala escura.</li>
		<li>Monitore a reação e remova a solução de reação quando a solução ficar brilhante para evitar manchas não específicas.</li>
		<li>Lave os ESCs duas vezes com 1x PBS.</li>
		<li>Evite que a amostra seque, cobrindo os ESCs com 1x PBS ou meio de montagem. NOTA: Uma mancha vermelha ou roxa aparecerá para a expressão AP. A placa pode ser armazenada em uma geladeira de 4 °C.</li>
	</ol></li>
	<li>RT-qPCR 
	<ol>
<li>Coletar células em vários estágios seguindo as etapas 1.6-1.7 e 2.4 para ESCs e EBs e NPCs, respectivamente.</li>
		<li>Isolar o RNA usando solução de RNA, DNA e extração de proteínas (ver Tabela de Materiais).</li>
		<li>Gerar cDNA com um kit de transcriptase reversa (ver a Tabela de Materiais) e seguir o manual do fabricante.</li>
	</ol></li>
	<li>Fixação e incorporação 
	<ol>
<li>Colher EBs e NPCs conforme descrito acima (passo 2.6) e corrigi-los com solução de paraformaldeído (PFA) de 4% em 1x PBS por 30 minutos à temperatura ambiente.</li>
		<li>Retire o PFA e lave a amostra com 1x PBS por 5 minutos.</li>
		<li>Coloque a amostra em uma diluição serial de 1x PBS, 10%-, 20%-, e 30% de soluções de sacarose a 25\u201228 °C onde a amostra é transferida para a próxima solução após 30 minutos de incubação. NOTA: A amostra pode ser armazenada na solução de sacarose de 30% a 4 °C antes de continuar com a etapa de incorporação.</li>
		<li>Molhe a ponta da pipeta com solução de sacarose antes de colocar a amostra (sem empilhá-las) no centro do molde crio-mold e pipeta para fora do excesso de líquido. NOTA: O papel do filtro também pode ser usado para remover a solução em excesso. Molhar a ponta da pipeta com solução de sacarose é importante para evitar que os EBs e NPCs grudem nas paredes da ponta.</li>
		<li>Adicione cuidadosamente a solução de temperatura de corte (OCT) ideal ao molde sem reutilizar as amostras e remova bolhas em excesso com uma pipeta.</li>
		<li>Coloque o molde com a amostra em uma batedeira de laboratório em baixa velocidade para agitar levemente a amostra por 15 minutos. Isso ajuda a resolver os EBs e NPCs até o fundo se eles forem resuspensados na solução OCT.</li>
		<li>Congele rapidamente a amostra colocando o molde em nitrogênio líquido ou em gelo seco. NOTA: As amostras podem ser armazenadas em um congelador de -70 °C antes de continuar para o próximo passo.</li>
	</ol></li>
	<li>Criocendo 
	<ol>
<li>Defina o criostat para esfriar para -20 a -18 °C antes de transferir a amostra para o instrumento.</li>
		<li>Retire o bloco OCT congelado do molde e fixe-o no suporte com uma pequena solução oct colocada na superfície do suporte.</li>
		<li>Alinhe o bloco OCT para que os EBs e NPCs estejam mais próximos da lâmina para garantir que a amostra não seja perdida durante a secção.</li>
		<li>Seção cuidadosa 10 μm do bloco e preste muita atenção às fatias que contêm a amostra.</li>
		<li>Coloque rapidamente a fatia OCT contendo a amostra no slide de vidro de incorporação de tecido e permita que as fatias de OCT sequem ao ar por 1 h à temperatura ambiente. NOTA: As amostras podem ser armazenadas a -70 °C para uso posterior.</li>
	</ol></li>
	<li>Imunofluorescência (IF) 
	<ol>
<li>Bloco de seções OCT ou câmaras de cultura contendo neurônios em soro de burro 10% normal/0,1% Triton X-100 em 1x PBS por 1 h à temperatura ambiente.</li>
		<li>Incubar as amostras em anticorpo primário diluído em 5% de soro de burro normal/0,05% Triton X-100 em 1x PBS durante a noite a 4 °C.</li>
		<li>Lave amostras em 1x PBS/0,1% Triton X-100 três vezes por 5 min cada lavagem.</li>
		<li>Incubar as amostras com anticorpo secundário diluído em 5% de soro de burro normal/0,05% Triton X-100 em 1x PBS por 1h à temperatura ambiente.</li>
		<li>Lave amostras em 1x PBS/0,1% Triton X-100 três vezes por 5 min cada lavagem e incubar as amostras em 1 μg/mL DAPI.</li>
		<li>Monte as amostras com um deslizamento de tampa e algum meio de montagem e deixe secar.</li>
		<li>Observe as amostras sob um microscópio de fluorescência.</li>
	</ol></li>
	<li>Análise de RNA-seq 
	<ol>
<li>Recolher as células em vários estágios e realizar a extração de RNA (ver passo 3.2).</li>
		<li>Preparar as bibliotecas cDNA, realizar sequenciamento profundo e análise de dados de acordo com o protocolo descrito em Wang et al.8.</li>
		<li>Execute a análise de Ontologia genética (GO) usando o pacote R, clusterProfiler.</li>
	</ol></li>
	<li>Citometria de fluxo 
	<ol>
<li>Colete ESCs seguindo as etapas 1.6-1.7 e resuspenque as células em médio. Colete os EBs e NPCs seguindo o passo 2.4 e resuspenque as células em médio.</li>
		<li>Filtre a suspensão celular usando o coador de células de nylon de 40 μm em um novo tubo cônico de 15 mL.</li>
		<li>Meça o sinal de GFP das amostras utilizando o citômetro de fluxo (realizado pela instalação central da instituição).</li>
	</ol></li>
</ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Akt/mTOR+pathway+in+cancer+stem/progenitor+cells+is+a+potential+therapeutic+target+for+glioblastoma+and+neuroblastoma.">The Akt/mTOR pathway in cancer stem/progenitor cells is a potential therapeutic target for glioblastoma and neuroblastoma.</a> Oncotarget. 9 (71), 33549-33561 (2018).</li><li>Bastiaens, A. J., 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=Advancing+a+MEMS-Based+3D+Cell+Culture+System+for+in+vitro+Neuro-Electrophysiological+Recordings.">Advancing a MEMS-Based 3D Cell Culture System for in vitro Neuro-Electrophysiological Recordings.</a> Frontiers in Mechanical Engineering. 4, 1-10 (2018).</li><li>Antill-O'Brien, N., Bourke, J., O'Connell, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-layer:+The+case+for+3D+bioprinting+neurons+to+create+patient-specific+epilepsy+models.">Layer-by-layer: The case for 3D bioprinting neurons to create patient-specific epilepsy models.</a> Materials. 12 (19), (2019).</li><li>Duval, 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=Modeling+physiological+events+in+2D+vs.+3D+cell+culture.">Modeling physiological events in 2D vs. 3D cell culture.</a> Physiology. 32 (4), 266-277 (2017).</li><li>Joshi, P., Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+imaging+(HCI)+on+miniaturized+three-dimensional+(3D)+cell+cultures.">High content imaging (HCI) on miniaturized three-dimensional (3D) cell cultures.</a> Biosensors. 5 (4), 768-790 (2015).</li></ol>

Os autores declaram que não há interesses financeiros concorrentes.

O método de diferenciação neural de células-tronco embrionárias do camundongo foi estabelecido há décadas e os pesquisadores continuaram a modificar os protocolos anteriores ou criar novos para vários propósitos7,10,11. Utilizamos uma série de ensaios para analisar de forma abrangente a eficiência e o progresso das etapas de diferenciação dos mESCs aos neurônios, que podem ser utilizados na análise de outras difer...

Desde sua primeira derivação da massa celular interna dos blastocistos do camundongo em desenvolvimento1,2, as células-tronco embrionárias do rato (mESC) têm sido usadas como ferramentas poderosas para estudar a autoconexão e diferenciação das células-tronco3. Além disso, estudar a diferenciação do MEESC leva a uma tremenda compreensão dos mecanismos moleculares que podem melhorar a eficiência e a segurança na terapia baseada em células-tronco no tratamento de doenças como distúrbios neurodegenerativos4. Em comparação com os modelos animais, este sistema in vitro oferece muitas vantagens, incluindo simplicidade na prática e avaliação, baixo custo na manutenção de linhas celulares em contraste com os animais e relativa facilidade em manipulações genéticas. No entanto, a eficiência e a qualidade dos tipos de células diferenciadas são frequentemente afetadas por diferentes linhas de mESCs, bem como pelos métodos de diferenciação5,6. Além disso, os ensaios tradicionais para avaliar a eficiência de diferenciação dependem do exame qualitativo de genes marcadores selecionados que carecem de robustez e, portanto, não conseguem compreender as mudanças globais na expressão genética.
Aqui pretendemos usar uma bateria de ensaios para avaliação sistemática da diferenciação neuronal. Utilizando ambas as análises in vitro tradicionais em marcadores selecionados e RNA-seq, estabelecemos uma plataforma para medição da eficiência de diferenciação, bem como as alterações transcriômicas durante este processo. Com base em um protocolo previamente estabelecido7,geramos corpos embrionários (EBs) através da técnica de queda suspensa, seguidos pela indução usando quantidade suprafisiológica de ácido retinóico (RA) para gerar células progenitoras neurais (NPCs), que foram posteriormente diferenciadas aos neurônios com meio de indução neural. Para examinar a eficiência da diferenciação, além dos ensaios tradicionais de RT-qPCR e imunofluorescência (IF), realizamos RNA-seq e citometria de fluxo. Essas análises fornecem uma medição abrangente da progressão da diferenciação específica do estágio.

<table border="0" cellpadding="0" cellspacing="0" style="width:805px;" width="804">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">0.05% Trypsin + 0.53mM EDTA 1X</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-052-CV</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">0.1% Gelatin</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">G1890-100G</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">16% Paraformaldehyde</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:127px;">28908</td>
			<td>Diluted in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">40-&#956;m cell strainer</td>
			<td style="width:148px;">Falcon</td>
			<td style="width:127px;">352340</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Albumax</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">11020021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">AlexaFluor 488 goat anti-mouse IgG (H+L)</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">A11001</td>
			<td>Antibody was diluted at 1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Alkaline Phosphatase Staining Kit II</td>
			<td style="width:148px;">Stemgent</td>
			<td style="width:127px;">00-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">AzuraQuant Green Fast qPCR Mix LoRox</td>
			<td style="width:148px;">Azura Genomics</td>
			<td style="width:127px;">AZ-2105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">B27 supplement</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">BD FACSCanto</td>
			<td style="width:148px;">BD</td>
			<td style="width:127px;">657338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">bFGF</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">11123149001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">BioAnalyzer High Sensitivity DNA Kit</td>
			<td style="width:148px;">Agilent</td>
			<td style="width:127px;">5067-4626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chir99021</td>
			<td style="width:148px;">Cayman Chemicals</td>
			<td style="width:127px;">13122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chloroform</td>
			<td style="width:148px;">C298-500</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">DAPI</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">R37606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">DMEM</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">DMEM/F12 medium</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">11320033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">EB buffer</td>
			<td style="width:148px;">Qiagen</td>
			<td style="width:127px;">19086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ethanol</td>
			<td style="width:148px;">111000200</td>
			<td style="width:127px;">Pharmco</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Fetal bovine serum</td>
			<td style="width:148px;">Atlanta Biologicals</td>
			<td style="width:127px;">S10250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Fisherbrand Superfrost Plus Microscope Slides</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:127px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">HiSeq 2500 Sequencing System</td>
			<td style="width:148px;">Illumina</td>
			<td style="width:127px;">SY-401-2501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Isopropanol</td>
			<td style="width:148px;">BDH1133-4LG</td>
			<td style="width:127px;">BDH VWR Analytical</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">L-glutamine</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">25030024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">LIF</td>
			<td style="width:148px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Collected from MEF supernatant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">m18srRNA primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-GCAATTATTCCCCATGAACG-3&#39; 
			5&#39;-GGCCTCACTAAACCATCCAA-3&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">MEM Non-essential amino acids</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mNanog primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGGCTTTGGAGACAGTGAGGTG-3&#39; 
			5&#39;-TGGGTAAGGGTGTTCAAGCACT-3&#39;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">mNes primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGTGCCCAGTTCTAGTGGTGTCC-3&#39; 
			5&#39;-CCTCTAAAATAGAGTGGTGAGGGTTG-3&#39;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">mNeuroD1 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-CGAGTCATGAGTGCCCAGCTTA-3&#39; 
			5&#39;-CCGGGAATAGTGAAACTGACGTG-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mOct4 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGATCACTCACATCGCCAATCA-3&#39; 
			5&#39;-CGCCGGTTACAGAACCATACTC-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mPax6 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-CTTGGGAAATCCGAGACAGA-3&#39; 
			5&#39;-CTAGCCAGGTTGCGAAGAAC-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">N2 supplement</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Nestin primary antibody</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:127px;">MAB5326</td>
			<td>Antibody was diluted at 1:200 for IF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Neural basal</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">21103049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Neurofilament primary antibody</td>
			<td style="width:148px;">DSHB</td>
			<td style="width:127px;">2H3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit</td>
			<td style="width:148px;">BioO Scientific</td>
			<td style="width:127px;">NOVA-5138-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">PD0325901</td>
			<td style="width:148px;">Cayman Chemicals</td>
			<td style="width:127px;">13034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Penicillin/streptomycin</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Phosphate-buffered saline (PBS)</td>
			<td style="width:148px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">- Potassium chloride</td>
			<td style="width:148px;">P217-500G</td>
			<td style="width:127px;">VWR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Potassium phosphate monobasic anhydrous</td>
			<td style="width:148px;">0781-500G</td>
			<td style="width:127px;">VWR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Sodium chloride</td>
			<td style="width:148px;">BP358-10</td>
			<td style="width:127px;">Fisher Bioreagents</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Sodium phosphate, dibasic, heptahydrate</td>
			<td style="width:148px;">SX0715-1</td>
			<td style="width:127px;">Milipore</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Random hexamer primer</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:127px;">SO142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Retinoic acid</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">R2625</td>
			<td>Prepared in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sodium pyruvate</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-000-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sucrose</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">84097</td>
			<td>Diluted in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">SuperScript III Reverse Transcriptase</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">18064022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:148px;">Sakura</td>
			<td style="width:127px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">TriPure Isolation Reagent</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:127px;">11667165001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">TruSeq Rapid</td>
			<td style="width:148px;">Illumina</td>
			<td style="width:127px;">20020616</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">&#946;-mercaptoethanol</td>
			<td style="width:148px;">Fisher BioReagents</td>
			<td style="width:127px;">BP176-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation and characterization of neural progenitors and neurons from mouse embryonic stem cells

Descrevemos o procedimento passo-a-passo para cultivar e diferenciar células-tronco embrionárias do camundongo em linhagens neuronais, seguido por uma série de ensaios para caracterizar as células diferenciadas. As células-tronco embrionárias do rato E14 foram usadas para formar corpos embrionários através do método de queda suspensa, e então induzidas a se diferenciar em células progenitoras neurais por ácido retinóico, e finalmente diferenciadas em neurônios. A reação em cadeia de polimerase de transcrição reversa quantitativa (RT-qPCR) e os experimentos de imunofluorescência revelaram que os progenitores e neurônios neurais exibem marcadores correspondentes (nestin para progenitores neurais e neurofilamento para neurônios) nos dias 8 e 12 pós-diferenciação, respectivamente. Experimentos de citometria de fluxo em uma linha E14 expressando um repórter gfp orientado pelo promotor Sox1 mostraram que cerca de 60% das células no dia 8 são GFP positivas, indicando a diferenciação bem sucedida das células progenitoras neurais nesta fase. Finalmente, a análise do RNA-seq foi utilizada para traçar o perfil das alterações transcriômicas globais. Esses métodos são úteis para analisar o envolvimento de genes e caminhos específicos na regulação da transição da identidade celular durante a diferenciação neuronal.

Como representação do nosso método, realizamos um experimento de EB, NPC e diferenciação de neurônios em células E14. As células E14 foram cultivadas em MEFs irradiados γ(Figura 1A)até que a população de MEF irradiada γ diluída. Confirmamos a pluripotência das células E14 realizando coloração de Fosfattase Alcalina (AP)(Figura 1B)e posteriormente RT-qPCR (veja abaixo) para marcadores Nanog e Oct4. As células E14 sem MEF γ fo...

Watch this Scientific Journal Video about Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells at JoVE.com

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

Descrevemos o procedimento para a diferenciação in vitro de células-tronco embrionárias do rato em células neuronais usando o método de queda de suspensão. Além disso, realizamos uma análise fenotípica abrangente através do RT-qPCR, imunofluorescência, RNA-seq e citometria de fluxo.

Diferenciação e caracterização de progenitores neurais e neurônios de células-tronco embrionárias do rato

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

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Research

JoVE Journal

Developmental Biology

4.7K visualizações.  Penn State Hershey Cancer Institute. Descrevemos o procedimento para a diferenciação in vitro de células-tronco embrionárias do rato em células neuronais usando o método de queda de suspensão. Além disso, realizamos uma análise fenotípica abrangente através do RT-qPCR, imunofluorescência, RNA-seq e citometria de fluxo.

Vídeo: Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Diferenciação Neural of Mouse Células-Tronco Embrionárias em soro livre de cultura em monocamada

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Biologia do Desenvolvimento

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Regulação epigenética da Cardiac diferenciação de células estaminais embrionárias e Tecidos

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Dirigido a diferenciação de progenitores hematopoiéticos primitivo e definitiva de células pluripotentes humanas

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Diferenciação eficiente dos Pluripotent Stem Cells para NKX6-1<sup&gt; +</sup&gt; Progenitores pancreáticas

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Análise do induzida por ácido retinóico Neural diferenciação de células estaminais embrionárias rato em corpos embrióides Dois e tridimensionais

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rápida, dirigida a diferenciação de células epiteliais de pigmento Retinal de células-tronco humanas embrionárias ou induzida de Pluripotent

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Isolamento, cultura e diferenciação de células do estroma da medula óssea e osteoclasto progenitores dos ratos

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Geração de células-tronco neurais induzidas de células mononucleares periféricas e diferenciação para precursores de neurónios dopaminérgicos para estudos de transplante

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Diferenciação neural eficiente usando a cultura unicelular de células-tronco embrionárias humanas

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

Preparação de Pequenas Bibliotecas de RNA para Sequenciamento de Embriões de Camundongos Primitivos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...