Kristen Brennand is a New York Stem Cell Foundation - Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.

1. hiPSC Diferenciação de células progenitoras neurais <ol><li> Crescer e expandir hiPSCs em células estaminais embrionárias humanas (HES) mídia (Tabela 1) co-cultivadas em um fibroblastos de rato embrionárias (MEF) camada alimentadora até grandes (mas subconfluentes) colônias estão prontos para a diferenciação neural através de um organismo embryoid (EB) intermediário (Figura 2). Condições de rotina cultura hiPSC estão bem descritos em outros lugares 16,17; resumidamente, hiPSCs crescer em meios de HES em uma camada de alimentação MEF até à confluência, em seguida passagem por via enzimática com colagenase (1 mg / ml em DMEM) e expandir cerca de 1: 3 a cada 7 dias. </li><li> Para preparar as placas revestidas com poli-L-ornitina / laminina, placas de revestimento com 10 g / ml de poli-L-ornitina em água estéril para as superfícies de plástico (50 g / ml para as superfícies de vidro) e incubar a temperatura ambiente durante 3-24 h. Lavar pelo menos uma vez com água estéril e, em seguida, cubra com 5g / ml Laminin em PBS estéril a TA durante 3-24 horas. NOTA: Se envolvido em plástico, as placas podem serarmazenados por até seis meses a -20 ºC. </li><li> No Dia 1, enzimaticamente levantar hiPSCs confluentes (geralmente cultivadas em MEFs, ou hiPSCs cultivadas em meios definidos como em Matrigel TeSR 18) a partir da placa de grandes colónias utilizando 1 mg / ml de colagenase IV em DMEM / F12. NOTA: após 1-2 horas de incubação a 37 ºC, as colônias serão flutuando no prato. </li><li> Delicadamente, lave colônias hiPSC (por sedimentação, não centrifugação) 1-2x com DMEM / F12, ressuspender em 2 mL / poço de N2 / media B27 (Tabela 1) e transferir para pratos não-aderente de 6 poços (combinar 3 poços em 1 -bem) 19. NOTA: Durante a noite, irá formar colônias flutuantes aglomerados esféricos denominados &quot;corpos embrióides&quot; (EBS). Esperar a morte celular substancial. </li><li> No dia 2, placas de inclinação e permitir EBS para resolver. Retire cuidadosamente a mídia e lavar uma vez com DMEM / F12 para remover detritos. Alimente com N2 / B27 meios suplementados com inibidores SMAD duplos (0,1M LDN193189 e 10M SB431542) 12 </sup&gt;. </li><li> Nos Dias 3-7, neuralization ocorrerá no contexto de dupla inibição SMAD; verificar que a EBS são redondos, mas não cística. Alimente EBs cada segundo dia com a mídia N2 / B27 suplementadas com 0,1 M LDN193189 e 10M SB431542. </li><li> Nos dias 7-14, a seguir ao plaqueamento de EBs poli-L-ornitina / laminina placas revestidas, veja utilizando um microscópio de campo claro que rosetas neurais começam a aparecer dentro de poucos dias (caracterizado como aglomerados redondos de células neuroepiteliais com polaridade APICO-basal; A Figura 1). Continuem a alimentar EBs aderentes a cada segundo dia, com media N2 / B27 suplementado com 0,1 M LDN193189, 10M SB431542 e 1 g / ml laminina. </li></ol> 2. Harvest of Neural Rosetas NOTA: Recomendamos que rosetas neurais ser enzymatically colhidas utilizando Reagente de Selecção Neural Rosette 20 ou reagente seleção similar. Embora rosetas neurais podem ser colhidos manualmente em 6 poços de poli-L-ornitina / laminina placa revestida, this metodologia leva treinamento intensivo para mestre, e, dependendo da habilidade do usuário, pode requerer uma segunda rodada de picking no dia 20 para enriquecer ainda mais para NPCs e empobrecem os tipos de células não-neuronais. <ol><li> Para selecção enzimática de rosetas neurais no dia 14, os meios de aspirado de EBs aderidas e adicionar 1 ml de reagente de selecção roseta neural por poço para uma placa de 6 poços. Incubar a 37 ºC durante 1 hora. </li><li> Com uma pipeta P1000, remover suavemente enzima de cada poço. Adicionar 1 ml de DMEM / F12 por poço para lavar. </li><li> Com um P1000, recolher o 1 ml de DMEM / F12 e rapidamente expelir o DMEM / F12 de volta para dentro do poço, separando assim as rosetas da placa. Recolha as rosetas em um tubo falcon. </li><li> Pipetar mais 1 ml de DMEM / F12 e rapidamente em expulsar o mesmo para separar bem rosetas restantes. (Não triturar. Tente não quebrar agregados roseta). Recolha as rosetas neurais em um mesmo tubo falcon. </li><li> Repita o passo 2.4, se necessário. Se rosetas não retirar prontamente, adicione more enzima e repetir o processo (passos 2,1-2,4) </li><li> Girar células a 300 xg durante 3 min. Aspirar a lavagem, e re-suspender rosetas em 2 ml de meio de NPC (Tabela 1). Transferência de células (1: 1) em um poli-L-ornitina / laminina revestido placa de 6 poços. </li><li> A partir Dias 15-21, as rosetas neurais irá expandir; rosetas de alimentação a cada segundo dia, com media NPC. </li><li> Avaliar a qualidade das rosetas neurais e assegurar que as células do tipo fibroblasto planas não estão a expandir dentro da cultura. NOTA: Se não-rosetas persistirem, a cultura NPC às vezes pode ser reaproveitado por picking o manual, selecionando apenas os melhores das rosetas escolhidos em Accutase. Dissociar 15 min a 37 ºC. Pellet, lavar e placa em meios NPC para 24 poços de poli-L-ornitina / laminina placa revestida. </li></ol> 3. Ampliação da Neural células progenitoras NOTA: hiPSC NPCs podem ser cultivadas em qualquer Matrigel- ou poli-L-ornitina / laminina placas revestidas. Normalmente usamos Matrigel-placas de como eles podem ser preparados de forma mais rápida e com menor custo. <ol><li> Para preparar as placas revestidas com Matrigel, descongelar rapidamente e ressuspender uma alíquota congelada de 1 mg em 24 ml de Matrigel frio DMEM / F12 e distribuir imediatamente 2 ml para cada poço de duas placas de seis poços. Incubar durante pelo menos 1 h a 37 ºC. NOTA: Matrigel necessita apenas de ser aspirado, não lavadas, imediatamente antes da utilização. </li><li> Alimente NPCs cada segundo dia e manter a densidade muito elevada ou eles vão diferenciar espontaneamente aos neurônios. NOTA: NPCs Embora mais estabelecidas são tipicamente divididas 1: 4 a cada semana, NPCs muito baixos de passagem podem ser divididas 1: 2 como com pouca frequência quanto uma vez a cada duas semanas. </li><li> Para dividir, media primeiros aspirado e adicione 1 ml quente Accutase (1x) por poço de 6 poços. Incubar a 37 ° C por 10-15 min. </li><li> Suavemente transferir células destacadas para um tubo de 15 ml contendo DMEM / F12 com tão pouco quanto possível a tensão mecânica. Não titulação em células enquanto enzima. Células de pellets por spinning a 1000 xg durante 5 min. </li><li> Aspirar a lavagem, e re-suspender NPCs em ~ 1 ml de meio por poço original NPC de 6 poços. NOTA: Esperar que um bem confluente de um de 6 poços prato tem 5-10.000.000 células; isto pode ser confirmado por contagem de uma aliquota de células 10l usando um hematocytometer antes da re-deposição. </li><li> Após re-suspensão, NPCs re-placa como NPCs, neurônios ou neurospheres. Para manter NPCs, placa aproximadamente 1-2 milhões de células por poço de uma placa de 6 poços. A eficiência de formação de neuroesferas podem variar entre experiências e as linhas celulares, mas em geral ocorre melhor se entre 200.000 e 1.000.000 células são semeadas por poço de um não-aderente placa de 6 poços; se ocorrer aglutinação das neuroesferas, reduzir o número de células semeadas. Para diferenciar a neurônios, placa de cerca de 200.000 células por poço de uma placa de 6 poços, substituindo media NPC com a mídia neurônio. </li><li> Immunohistochemically validar cada linha NPC estabelecida. Uma vez que o material celular suficiente tenha sido expanded, NPCs de etiqueta com anticorpos para Nestin e SOX2. Linhas Validated NPC também devem ser diferenciadas em neurônios durante 4-6 semanas, e marcadas com anticorpos para III-tubulina e MAP2AB. <ol><li> Fixar as células em 4% de paraformaldeído em PBS a 4C durante 10 min. Permeabilizar NPCs à TA durante 15 min em 1,0% de Triton em PBS e, em seguida bloquear em 5% de soro de burro com 0,1% de Triton à TA durante 30 min. </li><li> Incubar com anticorpo primário em 5% de soro de burro com 0,1% de Triton, durante a noite a 4C. NOTA: Recomendamos os seguintes anticorpos: cabra anti-Sox2, 1: 200; nestina de murganho anti-humano, 1: 200; de coelho anti-βIII-tubulina, 1: 500; de ratinho anti-βIII-tubulina, 1: 500; de ratinho anti-MAP2AB, 1: 200. (Figura 2). </li><li> Após uma lavagem com PBS, as células incubar com anticorpos secundários conjugados com o anticorpo secundário adequado para cabra, ratinho ou coelho a 1: 300, em em 5% de soro de burro com 0,1% de Triton durante 1-2 horas à temperatura ambiente. Para visualizar os núcleos, células mancha com 0,5 ug / ml de DAPI (4 &#39;, 6-diamidino-2-fenilindol) e, em seguida, montar com Vectashield. </li></ol></li></ol> 4. NPC Transdução <ol><li> Use spinfection (centrifugação das placas de cultura de células na presença do vírus a 1.000 xg durante 1 hora) para aumentar a percentagem de células transfectadas 21. Aspirar media e substituir com a superexpressão relevante (ou controle) lentivírus (ou retrovírus), titulado para a multiplicidade desejada de infecção (MOI) (normalmente 1-10), diluída em meio NPC. Utilizar 1,5 mL de volume por poço de uma placa de 6 poços (ou um volume de escala apropriada para outros tipos de placas de cultura de tecido). Giram em 1000 xg e 37 ºC durante 1 hora em uma placa de centrífuga. NOTA: O ideal é que a transdução de NPCs com lentivírus ou vetores retrovirais dentro de 1-2 dias de divisão. Seja usando vírus comerciais ou obtidos em laboratório, pode ser útil para titular adequadamente lotes de vírus com antecedência por diluição em série. (Figura 3). </li><li> Para reduzir celmorte lular, substituir a mídia dentro de 8 horas de spinfection. NOTA: a integração virai e a expressão do transgene deve ser detectável dentro de 24 horas por meio de fluorescência de gene repórter. (Se o vírus não tem um repórter fluorescente, isso é mais difícil de avaliar; transfecção de sucesso pode ser melhor validada por immunoblot, imuno-histoquímica ou qPCR para produtos superexpressão.) Expressão completa pode demorar 3-7 dias; spinfections repetidas com vírus adicional pode ser necessário para aumentar a percentagem de células transfectadas. Spinfections recorrentes podem ocorrer diariamente, mas não precisa ser tão freqüente. Idealmente, se estiver usando um repórter fluorescente,&gt; 80% das células devem ser rotulados. </li></ol> 5. Neurosphere Migration Assay NOTA: As neuroesferas forma espontaneamente, após a dissociação enzimática de NPCs (por uma maneira idêntica à utilizada na expansão NPC - passos de 3,3-3,6), se as células são cultivadas em suspensão em meio de NPC. <ol><li> Em resumo, crescem dissociarNPCs d em media NPC para 48 horas em placas não aderentes, a fim de gerar neurospheres. (Figura 4). </li><li> Para a migração neuroesfera, para preparar as placas de Matrigel frescos usando meios NPC frio, em vez de DMEM, numa placa de 96 poços, 1-2 h antes da colheita de neuroesferas. Não aspirar Matrigel dos poços. </li><li> Conforme publicado originalmente para estaminais embrionárias derivadas de células neurospheres 22, escolher manualmente neurospheres NPC-derivados sob um microscópio, depois de lavar uma vez na mídia NPC para remover os restos celulares. Transferir uma neuroesfera a cada poço de uma placa revestida Matrigel de 96 poços. </li><li> Adicionar mais 0,5 mg adicionais de Matrigel, diluiu-se em meios frios NPC, para as neuroesferas em cada placa de 96 poços. </li><li> Centrar cada manualmente neuroesfera individual no meio da cavidade utilizando uma ponta de pipeta. Nota: É importante escolher neurospheres de tamanho semelhante, a fim de reduzir a variabilidade nos resultados. </li><li> Permitir que a migração ocorra durante 48 horas e, em seguida, corrigir células wom paraformaldeído 4% e mancha com marcadores imunoistoquímicos desejados. </li><li> Se a migração radial deve ser determinado, neurospheres fotografia em sua inteiramente usando um objetivo 4x microscópio. Excluir qualquer neuroesferas contacto com a borda do poço ou uma segunda neuroesfera a partir da análise. NOTA: Se ocorrer a migração por mais de 48 horas, a migração radial resultante provavelmente vai ser muito grande para a imagem usando uma objetiva de 4x. </li><li> Medir a migração média de cada neuroesfera usando software NIH ImageJ (Figura 5). Isto pode ser feito usando qualquer um dos métodos de análise descritos abaixo: <ol><li> Migração radial: <ol><li> Traçar manualmente a aresta da migração de células usando a ferramenta de seleção à mão livre, em seguida, usar a função Measure (Ctrl + M) para calcular a área da forma resultante. Antes de medir a área de certificar-se de que a área está marcada na janela Definir Medidas encontrada na guia Analisar. Use avalor da medição da área e a equação para a área de um círculo (A = πr 2) para determinar o raio (exterior). </li><li> Seguir a borda da neuroesfera original, medir a área e calcular o raio (interior) da mesma maneira. Calcular o total de migração radial como a diferença entre os raios interno e externo. </li></ol></li><li> A grande migração celular: <ol><li> Meça a distância percorrida dos cinco células mais afastadas da borda da neurosphere interior usando a ferramenta de seleção linear seguido pela Medida função (Ctrl + M). </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

Nós descrevemos métodos pelos quais a diferenciar hiPSCs em NPCs, um tipo de célula neural em que uma fração significativa da assinatura gene de neurônios hiPSC derivados é conservada e que pode servir como um proxy para as vias de desenvolvimento potencialmente contribuem para a patogênese da doença 8, 11. Além disso, como já detalhado, NPCs são uma população neural robustamente replicativo e facilmente transduzidas, que acreditamos que pode ser adequado para estudos moleculares e bioquímicos ...

Estudos de neurônios diferenciadas in vitro a partir de humanos células estaminais pluripotentes induzidas (hiPSCs) por nós 1 e outros 2,3 de expressão de genes indicam que os neurônios hiPSC assemelhar fetal em vez de adulto tecido cerebral. Actualmente, os modelos baseados em hiPSC pode ser mais adequado para o estudo da predisposição para, em vez de funções tardias da doença, neurológica. Nós já relataram que uma fracção significativa do gene assinatura de esquizofrenia neurónios derivados de hiPSC é conservado em células derivadas de esquizofrenia hiPSC progenitoras neurais (NPCs), indicando que NPCs pode ser um tipo de células útil para o estudo das vias moleculares que contribui para uma esquizofrenia . Nós e outros relataram migração aberrante, aumento do estresse oxidativo e espécies reativas de oxigênio, a sensibilidade a estresses ambientais sub-limiar e função mitocondrial prejudicada na esquizofrenia hiPSC NPCs 1,4-6, bem como a diminuição neuronal conectividade e função sináptica em neurônios esquizofrenia hiPSC 5,7-10. Se os factores moleculares que contribuem para a migração aberrante e / ou stress oxidativo na esquizofrenia hiPSC NPCs também estão na base da conectividade neuronal reduzido na esquizofrenia neurónios derivados de hiPSC, NPCs poderia ser uma população neural robusto e altamente replicativo com que para estudar os mecanismos responsáveis ​​pela doença. Além disso, porque pode-se gerar rapidamente grandes números de células e não precisam de esperar semanas ou meses para maturação neuronal, ensaios baseados em NPC são adequados para o estudo de coortes de pacientes e maiores são mais passíveis de rastreio de alto rendimento. Acreditamos que hiPSC NPCs pode servir como um proxy para as vias de desenvolvimento potencialmente contribuem para a patogênese da doença, como já foi demonstrado em desordens tão diversas como a esquizofrenia 1 e doença de Huntington doença 11. Para diferenciar NPCs hiPSCs, neural inicial emprodução é conseguida através da inibição de dupla SMAD (0,1 mM e 10 mM LDN193189 SB431542) 12. Antagonizando BMP e TGF sinalização com estas pequenas moléculas, endoderme e mesoderme especificação é bloqueado, acelerar a diferenciação neuronal e levando à formação de rosetas neurais visíveis dentro de uma semana de chapeamento. Padronização neural ocorre no início deste processo, presumivelmente, no período de formação de rosetas neural e imediatamente a seguir. Na ausência de outros sinais, estas células neurais primitivas assumir um destino prosencéfalo como anterior-13. Imediatamente após a formação de rosetas neural, e contínuo ao longo da expansão NPC, NPCs frontais do cérebro são cultivadas com FGF2 8,14. Eles têm potencial linhagem dupla e podem ser diferenciadas de populações neurais de 70-80% neurónios III-tubulina-positivos e 20-30% de proteína glial fibrilar ácida (GFAP) -positivas astrócitos (Figura 1). A maioria dos neurônios do cérebro anterior hiPSC são VGLUT1-positivo, E por isso são presumivelmente glutamatérgica e aproximadamente 30% dos neurónios estão GAD67-positivo (GABAérgica) 8. NPCs são rotineiramente passadas mais do que dez vezes in vitro, mantendo ao mesmo tempo perfis de diferenciação consistentes, e sem acumulação de anormalidades do cariótipo. Grupos têm relatado NPCs Passaging mais de 40 vezes 15, no entanto, descobrimos que mais de dez passagens, NPCs mostram aumento da propensão para a diferenciação de astrócitos. NPCs bem tolerar múltiplas Freeze-descongela e pode ser transferida para crescer como neurospheres simplesmente o cultivo em placas não aderentes. NPCs são eficientemente transduzidas por vectores virais, permitindo uma rápida avaliação das consequências moleculares e celulares da perturbação genética, e facilmente expansível para se obter material suficiente para estudos bioquímicos. Além disso, porque os vectores virais permitir robusta sobre-expressão e / ou eliminação de genes relevantes a doença, em qualquer controlo ou paciente derivado neural células, pode-se usar esta plataforma para testar o efeito de fundo genético nessas manipulações. Apesar de não ser adequado para sináptica ou ensaios baseados em atividades que exigem neurônios maduros, NPCs pode ser uma alternativa prática para muitas análises moleculares e bioquímicas diretas de células neurais derivadas de pacientes.

<table border="0" cellpadding="0" cellspacing="0" width="1174">
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			<td style="width:216px;">#11330</td>
			<td>for HES media</td>
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			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10565</td>
			<td style="width:500px;">for neural media</td>
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			<td height="21" style="height:21px;width:288px;">KO-Serum Replacement</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10828</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td height="21" style="height:21px;width:288px;">Glutamax</td>
			<td style="width:171px;">Life Technologies</td>
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			<td height="21" style="height:21px;width:288px;">2&#8208;mercaptoethanol (55mM 1000x)</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#21985-023</td>
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			<td height="21" style="height:21px;width:288px;">N2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#17502-048</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td height="21" style="height:21px;width:288px;">B27-RA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#12587-010</td>
			<td style="width:500px;">Needs to be lot tested</td>
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			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#13256-029</td>
			<td style="width:500px;">Resuspend in PBS + 1% BSA</td>
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			<td height="21" style="height:21px;width:288px;">LDN193189</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0074</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">SB431542</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0010</td>
			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">#450-02</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">GDNF&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">&#160;#450-10</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
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			<td height="21" style="height:21px;width:288px;">Dibutyryl cyclic-AMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">&#160;#D0627</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:288px;">L-ascorbic acid</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">#A0278</td>
			<td style="width:500px;">Resuspend in H20</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:288px;">STEMdiff Neural Rosette Selection Reagent</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td style="width:216px;">&#160;#05832</td>
			<td style="width:500px;"></td>
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			<td height="42" style="height:42px;width:288px;">Accutase</td>
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			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">Collagenase IV</td>
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			<td style="width:171px;">Millipore</td>
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			<td height="21" style="height:21px;width:288px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">P3655</td>
			<td style="width:500px;"></td>
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			<td height="21" style="height:21px;width:288px;">Laminin, Natural Mouse 1mg</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#23017-015</td>
			<td style="width:500px;"></td>
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			<td height="42" style="height:42px;width:288px;">BD Matrigel</td>
			<td style="width:171px;">BD</td>
			<td style="width:216px;">#354230</td>
			<td style="width:500px;">Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Tissue culture treated 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3506</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Ultra low attachment 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3471</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">goat anti-Sox2&#160;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td style="width:216px;">sc&#173;17320</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-human Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">MAB5326</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">rabbit anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">PRB&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">MMS&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-MAP2AB</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">M1406</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Plate centrifuge</td>
			<td style="width:171px;">Beckman Coulter</td>
			<td style="width:216px;">Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td>
			<td style="width:500px;"></td>
		</tr>
	</tbody>
</table>

a guide to generating and using hipsc derived npcs for the study of neurological diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

Rosetas neurais podem ser identificadas morfologicamente, utilizando um microscópio de campo claro, pela sua aparência característica como aglomerados redondos de células neuroepiteliais com polaridade APICO-basal (Figura 1). Embora NPCs são tipicamente cultivadas a uma densidade celular muito alta, imediatamente após a passagem em, ligeiramente soma em forma piramidal, e estrutura de neurites bipolar é visível (Figura 1D). NPCs validados expressam nestina e SOX2 na maioria das ...

Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Um Guia para a geração e uso hiPSC NPCs derivados para o Estudo das Doenças Neurológicas

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

a-guide-to-generating-using-hipsc-derived-npcs-for-study-neurological

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18.1K visualizações.  Icahn School of Medicine at Mount Sinai. This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Vídeo: A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Método para Desenvolvimento de Drogas Anti-Câncer Novel utilizando explantes Tumor de espécimes cirúrgicos


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Medicina

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizando Estimulação Magnética Transcraniana para o Estudo do sistema neuromuscular humano

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos Linhas tumor neuroendócrino de células como um modelo tridimensional para o estudo da terapia de tumores humanos Neuroendócrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolamento e Imortalidade de linhas celulares derivadas do paciente a partir de biópsia muscular para a doença Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Usando Saccadometry com Estimulação Cerebral Profunda para estudar normal e patológico função cerebral

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Um regime de fenotipagem de camundongos geneticamente modificados usados ​​para estudar genes implicados nas doenças humanas do envelhecimento

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Setting Up a Stroke Team Algorithm and Conducting Simulation-based Training in the Emergency Department - A Practical Guide

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can be&#160;achieved by either&#160;thrombolysis with recombinant tissue-plasminogen activator (rt-PA), the standard therapy for stroke patients who present within the first hours of symptom onset without contraindications, or by an endovascular approach, if a proximal brain vessel occlusion is detected. As the efficacy of both therapies declines over time, every minute saved along the way will improve the patient&#39;s outcome.
This critical situation requires thorough work and precise communication with the patient, the family and colleagues from different professions to acquire all relevant information and reach the right decision while carefully monitoring the patient. This is a high fidelity situation. In nonmedical high-fidelity environments such as aviation, Crew Resource Management (CRM) is used to enhance safety and team efficiency.
This guide shows how a Stroke Team algorithm, which is transferable to other hospital settings, was established and how regular simulation-based trainings were performed. It requires determination and endurance to maintain these time-consuming simulation trainings on a regular basis over the course of time. However, the resulting improvement of team spirit and excellent door-to-needle times will benefit both the patients and the work environment in any hospital.
A dedicated Stroke Team of 7 persons who are notified 24/7 by a collective call via speed dial and run a binding algorithm that takes approximately 20 min, was established. To train everybody involved in this algorithm, a simulation-based team training for all new Stroke Team members was conceived and conducted at monthly intervals. This led to a relevant and sustained reduction of the mean door-to-needle time to 25 min, and enhanced the feeling of stroke readiness especially in junior doctors and nurses.

Every min counts in acute stroke care. This guide shows how to establish a Stroke Team algorithm and enhance its performance with regular simulation training. The principles of Crew Resource Management (CRM) facilitate a straight workflow, reduce door-to-needle times and increase staff satisfaction.

Configurando um curso equipe Algoritmo e conduzir treinamento baseado em simulação no Departamento de Emergência - Um Guia Prático

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Automotive Radiochemical Synthesis of [<sup&gt; 18</sup&gt; F] 3F4AP: Um novo marcador de PET para imagens de doenças desmielinizantes

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Utilizando imagens da retina para estudar a demência

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...