Este trabalho foi apoiado pela Fundação Fritz Thyssen (Az.10.14.2.152). Somos gratos à facilidade de incorporação do tecido e da instalação do núcleo microscópio de CMMC. Estamos gratos pelas discussões e suporte técnico fornecido pelos membros do Laboratório de centrossoma e citoesqueleto Biology. Agradecemos Li Ming Gooi pela revisão do manuscrito.

1. Geração de Cérebro Organóides (23 dias) <ol><li> Iniciação de neuroectoderme (5 dias) Nota: Os seguintes pontos devem ser considerados antes do início da diferenciação. O método de reprogramação (lentiviral- etc, à base de microARN Sendai-a vírus, episomal-, OR) para obter iPSCs humanos devem, idealmente, ser o mesmo para todos os pacientes e controlar linhas IPSC. Vários kits de reprogramação e instruções baseadas em protocolos publicados estão disponíveis 15, 16, 17, 18. A qualidade das linhas de IPSC humanos é a chave para realizar uma diferenciação óptima. Monitorar colónia e morfologia das células com um microscópio e validar pluripotência, testando a expressão de marcadores, tais como OCT3 / 4, Nanog, ou TRA-1-60. <ol><li> iPSCs cultura humanos sob condições isentas de alimentação em meio A em um prato revestido com Engelbreth-Holm-Swarm(EHS) matriz. NOTA: Cresça iPSCs humanos e para manter uma condição de cultura definido e para evitar um passo adicional para a remoção de células de alimentação de rato embrionários (MEFs) antes do início da diferenciação sem soro livre de alimentador. O número de passagem óptima para iPSCs humanos para iniciar gamas de diferenciação a partir da passagem 15 em cima de reprogramação a passagem 70, no total. Quando passaging, separar colónias hiPSC como células agregação utilizando uma solução de separação celular apropriado com baixo esforço mecânico e uma pipeta serológica 2-mL para transferir agregados a um novo prato. Evitar a dissociação de células individuais, como este pode induzir a diferenciação e apoptose na maioria das linhas IPSC. Foi relatado que a longo prazo, a passagem de célula única poderiam aumentar alterações genómicas em iPSCs humano 19, 20. Evitar procedimentos de destacamento das células, as quais requerem um passo de centrifugação para remover a solução de desprendimento, pois isso reduz a viabilidade global de iPSCs. Testetodas as culturas para contaminações microbianas, particularmente micoplasma, em uma base regular, pois isso pode alterar a qualidade das iPSCs e sua capacidade de diferenciação. <ol><li> O revestimento de um tecido 60 milímetros prato de cultura com matriz EHS de acordo com as instruções do fabricante. </li><li> Descongelar uma aliquota de 1 x 10 6 iPSCs humanos. Semear as iPSCs em uma placa de cultura de tecidos de 60 mm revestidas com matriz EHS e contendo 5 mL de meio A (ver a Tabela Materiais). Alterar a forma e diariamente após a passagem de 5 a 7 dias, quando as células atingem ~ 80% de conflucia. NOTA: Passagem das iPSCs descongeladas a 80% de confluência utilizando métodos padrão, tais como o descolamento isenta de enzima de colónias de 21, pelo menos uma vez antes de iniciar a diferenciação. Em breve, remover o meio CIPS, lave as células uma vez com pré-aquecida até 37 Meio de Eagle Modificado de Dulbecco ° C: Mistura de Nutrientes F12 (DMEM / F12), e incuba-se as iPSCs seguindo o fabricante deinstruções usando o reagente A (ver a tabela de materiais). Não exceda o tempo de incubação recomendado para evitar a dissociação de células individuais. iPSCs humanos deve ser destacado e que flutua na forma de agregados, as células não é tão simples. Eles podem então ser transferidas para novas placas revestidas com matriz de EHS; por exemplo, iPSC agrega a partir de um prato de 60 mm pode ser distribuído a 4 novos pratos de 60 mm. </li><li> Verifique para micoplasma com um kit de detecção de micoplasma de acordo com as instruções do fabricante. Use iPSCs única isentos de micoplasma, como micoplasma pode alterar a capacidade de diferenciação iPSCs. </li></ol></li><li> Dissociar iPSCs (80% confluentes) e preparar uma suspensão de célula única usando o reagente B. <ol><li> Lavam-se as iPSCs uma vez com pré-aquecido (37 ° C) de DMEM / F12. </li><li> Adicionar pré-aquecido reagente B (por exemplo, 1 ml de uma placa de 60 mm) e incuba-se as iPSCs durante 5 min a 37 ° C e 5% de CO 2. </li><li> Flick 20 vezes com a ponta dos dedos sobre o lado inferior e deo prato para separar as iPSCs. Entrada para o destacamento de células sob o microscópio. </li><li> Pipetar a suspensão de células para cima e para baixo no prato 5 vezes com uma micropipeta 1 ml. </li><li> Adicionar 3 ml de meio A para diluir 1 mL de reagente B e recolher a suspensão celular num tubo de centrífuga de 15 mL. </li><li> Suavemente girar para baixo as iPSCs (500 xg) durante 4 min temperatura ambiente. </li><li> Ressuspender o sedimento celular em 1 ml de meio B e contar o número de células com um hemocitómetro. NOTA: Lembre-se de utilizar apenas meio B para a ressuspensão. Evitar o uso de meio A, uma vez que contém uma muito alta concentração de bFGF, que pode inibir a diferenciação. </li></ol></li><li> Dilui-se a suspensão de células a 4,5 x 10 5 culas por ml em meio B suplementado com 10 ^ M-rho associado inibidor de proteína cinase (Y-27632). </li><li> Adicionar 100 por po de uma forma não-aderente, de fundo em v, placa de 96 poços. NOTA: Certifique-se as células são igualmente distribuídos no SUSpensão por agitação do tubo de cada vez para retirar porções de 100 uL. É importante que cada cavidade deve conter um número de células equivalente, a fim de obter neuroesferas homogéneas em tamanho e forma (redondos, superfícies definidas). </li><li> Suavemente girar para baixo a placa com as células a 500 xg e a temperatura ambiente durante 3 min e incubar a 37 ° C e 5% de CO 2. </li><li> Mudar a forma diariamente através da remoção de 50 mL e adicionar 50 mL de meio fresco B em cada cavidade para os próximos 5 dias. </li></ol></li></ol> 2. Incorporação de neuroesferas em EHS Matriz (4 dias) <ol><li> Preparar o meio neuroesfera por mistura do seguinte: uma mistura de DMEM / F12 e meio C (v / v), 1:: 200 (v / v) suplemento 1, 1: 1 100 (v / v) suplemento 2 sem (w / o) A vitamina A, 1: 100 de L-glutamina, 0,05 mM de aminoácidos não essenciais (MEM), 100 U / mL de penicilina, 100 ug / ml de estreptomicina, 1,6 g / l de insulina, e mM β-mercaptoetanol 0,05. </li><li> Recolher o wit neurospheresha 200 mL micropipeta com uma ponta ~ 2 milímetros previamente cortado com tesoura estéreis. </li><li> Coloque as neuroesferas aproximadamente 5 mm de distância umas das outras no filme de parafina (3 x 3 cm2) numa placa vazia 100 mm e remover cuidadosamente tanto do meio restante quanto possível. </li><li> Adicionar uma gota (7 uL) de matriz para cada EHS único neuroesfera. </li><li> Incubar a matriz EHS gotas com as neuroesferas durante 15 minutos em uma incubadora. </li><li> Lavam-se as neuroesferas cuidadosamente a partir da película de parafina por rubor-los com meio neuroesfera. Para lavar, usar uma micropipeta 1 ml e um novo 100 milímetros placa de Petri contendo 10 ml de meio de neuroesfera. </li><li> Incubar as neuroesferas para os 4 dias seguintes e adicionar 2 mL de meio fresco neuroesfera no dia 2. NOTA: Certifique-se de que as prateleiras na incubadora são planas para que os neurospheres incorporado de matriz EHS não vai se aglutinarem em um lado do prato. </li></ol> 3. Organóides numa suspensão rotativaCultura (14 dias) <ol><li> Prepare cérebro médio organï¿½de por mistura do seguinte: uma mistura de DMEM / F12 e meio C (v / v), 1:: 200 (v / v) suplemento 1, 1: 100 (v / v) suplemento de 2 W / o um A vitamina A, 1: 100 de L-glutamina, MEM 0,05 mM, 100 U / mL de penicilina, 100 ug / ml de estreptomicina, 1,6 g / L de insulina, 0,5 uM dorsomorphin, 5 uM SB431542, e mM β-mercaptoetanol 0,05. </li><li> Adicionar 100 mL de cérebro organï¿½de meio a cada bal rotativo através dos seus braços laterais e colocá-los em uma incubadora para o pré-aquecimento durante pelo menos 20 min. </li><li> Defina-se um programa de agitação a 25 rpm, de acordo com as instruções do fabricante. NOTA: Antes de transferir os neurospheres incorporado de matriz EHS em frascos spinner, certifique-se que todos eles estão separados. Se dois ou mais estão ligados através da matriz de EHS, separá-los por corte da matriz de ligação com um bisturi. </li><li> transferir cuidadosamente as neuroesferas incorporado-matriz EHS em frascos de agitação contendo 100 mL de meio nos organï¿½deing uma pipeta serológica 2 mL. Utilizar os braços laterais do balão rotativo para transferir as neuroesferas para o balão. </li><li> Colocar os frascos rotativos numa plataforma de agitação magnética numa incubadora a 37 ° C e 5% de CO 2; este é dia 0 da cultura organóides. </li><li> Alterar o meio de uma vez por semana (ou mais frequentemente quando há uma mudança de cor), removendo metade do meio e adicionando a mesma quantidade de meio fresco. NOTA: Quando tirar os frascos spinner fora da incubadora, espere 3-5 minutos para permitir que os Organóides afundar até o fundo do frasco. Remover o meio, colocando a ponta da pipeta de vidro (ligada a uma bomba) sobre a superfície do líquido; Aspirar o meio cuidadosamente através de uma abertura lateral / braço do balão. Estas manipulações deve ser feito sob a capa laminar. </li></ol> 4. Análise do cérebro Organóides <ol><li> Fixação de Organóides <ol><li> Recolher os Organóides no dia 14 de cultura o wi bal rotativoth um corte de 1 mL micropipeta (corte a 5 mm). Coloque todos eles em um prato de 60 mm, e lavá-los uma vez com 5 mL de DMEM morno / F12, durante 3 min. </li><li> Preparar um tubo de 1,5 mL com 500 uL de paraformaldeído a quente 4% (PFA). Cuidado: use pele e proteção para os olhos e trabalhar sob um capuz de segurança ao manusear PFA fixador. </li><li> Colocar cada organï¿½de separadamente em cada tubo e corrigi-los por pelo menos 30 min à temperatura ambiente. Não corrigir os Organóides por mais de 60 min. Para mover os Organóides, utilizar uma ansa de inoculação ou qualquer outro conceber que é conveniente. </li><li> Remover o PFA e lavar os Organóides fixas duas vezes durante 10 minutos cada um com 1 mL de PBS. </li><li> Armazenar os Organóides em 1 ml de PBS a 4 ° C durante até 7 dias, até à sua utilização. </li></ol></li><li> Incorporar os Organóides para cryosectioning <ol><li> Remover o PBS e adicionar 1 ml de sacarose a 30% em solução de água destilada por cada tubo para desidratar os Organóides antes cryofreezing-los; após a adição sucrosolução si, os Organóides deve ser flutuante na superfície. Armazenar os Organóides durante a noite em solução de sacarose a 4 ° C; no dia seguinte, as Organóides deve ter afundado até o fundo do tubo. NOTA: Organóides pode ser armazenado durante até 3-5 dias a 4 ° C numa solução de sacarose, se necessário. </li><li> Encher um molde de espécime de vinilo com 400 uL de temperatura de corte óptima composto (OCT) e utilizar uma ansa de inoculação a colocar um organóide no centro do molde. Rotular o aro do molde com o nome da amostra. </li><li> Congelar o molde contendo organï¿½de-se a -80 ° C até cryosectioning. </li><li> cryoslides vidro do revestimento com 0,1% de solução de lisina de poli-L-(PLL) em PBS durante 5 min à temperatura ambiente e deixar secar as lâminas durante 3 h. Armazenar os slides a 4 ° C e aquecê-los até temperado ambiente antes do uso. NOTA: PLL-revestimento é um passo importante, uma vez que irá evitar que as fatias de organï¿½de flutuando para longe. Recolha e armazenamento da solução de PLL, a 4 ° C durante até 3meses. Antes de reutilização, filtra-se com uma seringa de 0,22 um e deixe aquecer até à temperatura ambiente. </li><li> Organóides secção cryofrozen em fatias 20-50 um de espessura em um vidro revestido de PLL-cryoslides 22. Deixe as lâminas com as secções secar durante 1 h à temperatura ambiente. Armazenar as secções a -80 ° C até processamento adicional. </li></ol></li></ol> 5. imunofluorescência Coloração de secções organóides NOTA: Para a caracterização geral de Organóides, coloração com a nestina, um marcador de células progenitoras neurais, e TUJ1, um marcador pan-neuronal, é recomendado. Como exemplos adicionais, a coloração imunofluorescente com fosfo-vimentina (p-Vim), os marcadores de células gliais radiais apicais mitóticos, e Arl13b, para cílio, são descritos. Para testar a apoptose, utilizar o ensaio terminal desoxinucleotidilo transferase (TdT) dUTP Nick-End Rotulagem (TUNEL). Colocar as lâminas em uma caixa de plástico durante as incubações para protegê-los da poeira, luz,e secar. <ol><li> Descongelar as lâminas durante 30 minutos à temperatura ambiente. </li><li> Lavar as lâminas duas vezes com 200 ul de PBS-glicina (0,225 g de glicina em PBS) durante 3 minutos para extinguir a autofluorescência induzida PFA. </li><li> Permeabilizar as secções com 200 uL de 0,5% de Triton X100 a 0,1% / tween em solução de PBS durante 10 min à temperatura ambiente. </li><li> Lavar duas vezes com 200 uL de solução de PBS-glicina durante 3 min. </li><li> Incubar deles com 200 uL de 0,5% de gelatina de peixe / 0,1% de Triton X100 em PBS durante 1 h à temperatura ambiente ou durante a noite a 4 ° C para bloquear a ligação não específica de antigénio. NOTA: Se um ensaio de TUNEL for necessário, realizar o ensaio de acordo com o protocolo do fabricante. Comece com o ensaio TUNEL antes de realizar a imunocoloração, como ele pode interferir com os anticorpos secundários e extinguir fluoróforos quando utilizado posteriormente. </li><li> Dilui-se os anticorpos em solução com as seguintes concentrações de bloqueio: nestina, 1: 200; p-Vim, 1: 500; TUJ1, 1: 200;Arl13b, 01:20; e anticorpos secundários, 1: 1.000. </li><li> Incubar com 200 uL do primeiro anticorpo primário (por exemplo, nestina) durante 1-2 h à temperatura ambiente ou durante a noite a 4 ° C. </li><li> Lavar 3 x 3 min com 200 ul de solução de bloqueio. </li><li> Dilui-se a primeira (anti-ratinho 488) Anticorpo secundário 1: 1000 em solução de bloqueio e incubar a corrediça em 200 ul durante 1-2 h à temperatura ambiente. De agora em diante, sempre proteger os slides da luz. </li><li> Lavar 3 x 3 min com 200 ul de solução de bloqueio. </li><li> Incubar com 200 uL do anticorpo primário ao lado (por exemplo, TUJ1) durante 1-2 h à temperatura ambiente ou durante a noite a 4 ° C. </li><li> Lavar 3 x 3 min com 200 ul de solução de bloqueio. </li><li> Adicionar 200 uL da próxima anticorpo secundário (anti-coelho de 647) durante 1-2 h à temperatura ambiente. </li><li> Lavar 3 x 3 min. com 200 ul de solução de bloqueio. </li><li> Adicionar 200 uL de 4&#39; , 6-diamidino-2-fenilindole (DAPI) a uma conc nM 30entration em PBS durante 15 min à temperatura ambiente para coloração nuclear. </li><li> Lavar 2 x 3 min com 200 ul de solução de bloqueio. </li><li> Lavar 1 x 1 min com 200 mL de água destilada e deixar as secções secar durante 10-20 minutos, até que nenhuma gota de água é óbvia visível mais. </li><li> Montar as secções com meio de inclusão. Armazená-los ao abrigo da luz a 4 ° C durante até várias semanas. </li><li> Prosseguir com a análise microscópica para a imagem uma visão geral da zona organóides, ventricular, placa cortical primitiva, e outras áreas de interesse. </li><li> Usar um microscópio confocal com um óleo de 63X filtros objectivos e fluorescentes, escolhido de acordo com os anticorpos secundários etiquetados com corante fluorescente utilizado 1, 10. </li></ol>

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+human+induced+pluripotent+stem+cells+follows+developmental+principles+but+with+variable+potency.">Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 4335-4340 (2010).</li><li>Wilson, P. G., Stice, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+differentiation+of+neural+rosettes+derived+from+human+embryonic+stem+cells.">Development and differentiation of neural rosettes derived from human embryonic stem cells.</a> Stem Cell Reviews. 2, 67-77 (2006).</li><li>Dhara, S. K., Stice, S. 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The authors declare that they have no competing financial interests.

MCPH é um distúrbio neurológico humano complexo que não pode ser recapitulado em modelos animais in vivo, ou em cultura de células humanas simples abordagens in vitro. A manifestação clínica da MCPH começa a aparecer durante o primeiro trimestre, quando no início neurogênese começa. Assim, Organóides cerebrais 3D representam um sistema experimental confiável para modelar desenvolvimento MCPH. Além disso, 3D Organóides cérebro humano são uma abordagem ideal uma vez que i) que permita a...

desordens do neurodesenvolvimento, tais como humanos, microcefalia, só pode ser pobremente estudado em modelos animais, devido ao facto de que os cérebros humanos têm uma superfície cortical estendida, uma característica única que difere a partir de animais não-humanos. Este aspecto torna o desenvolvimento do cérebro humano de um processo complexo que não pode ser suficientemente estudado num 2D, no sistema de cultura de células in vitro. Emergentes técnicas de cultura 3D permitem a geração de Organóides de tecido semelhante a partir de células estaminais pluripotentes induzidas (IPSCs). A diferenciação in vitro de células estaminais pluripotentes numa cultura em suspensão em 3D permite a formação de vários tipos de células de uma maneira atempada e específico de região, dando origem a, um tecido organizado estratificada 1, 2, 3. Graças a laboratórios que foram pioneiros tecnologias de cultura 3D e desmistificadas a complexidade da formação de órgãos, começando a partir de células estaminais,foi desenvolvido um método robusto de gerar Organóides cerebrais para delinear eventos precoces do desenvolvimento do cérebro humano e para modelar microcefalia in vitro 1, 2, 3. É digno de nota que nós adaptamos o método original desenvolvido por Lancaster et al. para gerar Organóides cerebrais 1. Este método foi modificado de acordo com as necessidades experimentais. O objectivo de um estudo de Gabriel et al. foi analisar os mecanismos celulares e moleculares da manutenção de células estaminais neurais durante o desenvolvimento do cérebro. A fim de fazer isso, um estudo mecanicista foi realizada através da análise de células progenitoras neurais (NPCs) em Organóides cerebrais 3D derivadas de um paciente microcefalia 4. Este paciente levou uma mutação em CPAP, uma proteína conservada centrosomal necessário para a biogénese centrossoma 5. Uma hipoglicemia amplamente aceitotese é que microcefalia é o resultado de um esgotamento do pool de NPC, e isto pode ser devido tanto a morte celular ou a diferenciação precoce 1, 6, 7, 8, 9. Ao analisar as zonas ventriculares (vzs) de Organóides cerebrais microcefalia, mostrou-se que um número significativo de NPCs sofrem divisão celular assimétrica, ao contrário Organóides cerebrais derivadas de um dador saudável 4. Análises microscópicas e bioquímicas extensas de Organóides cerebrais microcéfalos revelou um papel inesperado para CPAP em cílios oportuna desmontagem 4. Especificamente, CPAP mutado está associada com a desmontagem cílio retardado e a re-entrada do ciclo celular retardada, levando à diferenciação precoce de NPCs 4. Estes resultados sugerem um papel para cílios em microcefalia e thenvolvimento eir durante a neurogênese e tamanho do cérebro de controle 10. A primeira parte deste protocolo é uma descrição de um método de três passos para gerar Organóides cerebrais homogéneas. Como mencionado anteriormente, o protocolo Lancaster original foi adaptado e modificado para se adequar a uma finalidade. Em primeiro lugar, iPSCs humanos são cultivados num estado livre de alimentador definido em Engelbreth-Holm-Swarm (EHS) matriz. Este passo evita as variações de culturas de culas estaminais pluripotentes dependente de alimentação. Neste protocolo, a indução de diferenciação neuronal para formar epitélio neural começa directamente a partir iPSCs. Por saltando a etapa de formação de corpos embrióides (EB), os rendimentos de diferenciação neuronais de uma maneira mais controlada e orientada. Esta abordagem limita a formação espontânea e não-dirigida de outras camadas de células germinais, tal como mesoderme e endoderme. Ao aplicar este protocolo, neuroesferas contendo rosetas neurais podem ser colhidas no dia 5 para EHS incorporação matriz e cultura de suspensão estacionária. O meio utilizado para o organóide terceiro passo do nosso protocolo é suplementado com dorsomorphin e SB431542. Dorsomorphin é um inibidor de molécula pequena de proteína morfogénica do osso (BMP), e SB431542 inibe a via do TGFp / activina / nodal sinalização. A combinação destes factores poderia promover a diferenciação neuronal de forma mais eficiente do que o ácido retinóico sozinho 11, 12, 13, 14. Em conjunto, estas alterações permitem que a geração reprodutível de Organóides cerebrais, com variações mínimas através Organóides. Importante, este método foi aplicado para gerar robustamente Organóides cerebrais microcéfalos de iPSCs paciente, que transportam mutaes nos genes que afectem centrossomas e dinâmica do ciclo celular. A segunda parte deste protocolo dá instruções para preparar brain Organóides para a análise e interpretação dos defeitos celulares em microcefalia. Isto inclui a fixação, cryosectioning, coloração de imunofluorescência, e análise microscópica confocal. Este protocolo irá fornecer ao leitor uma descrição detalhada dos resultados previstos e com orientação para interpretação.

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-mouse 488</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">A-11001</td>
			<td style="width:212px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-rabbit 647</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">A-21245</td>
			<td style="width:212px;">Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arl13b</td>
			<td style="width:123px;">proteintech</td>
			<td style="width:119px;">17711-1-AP</td>
			<td style="width:212px;">ARL13B rabbit polyclonal antibody&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CELLSPIN system</td>
			<td style="width:123px;">IBS Integra Bioscience</td>
			<td style="width:119px;">183001</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DAPI</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">32670</td>
			<td style="width:212px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM/F-12</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">31331093</td>
			<td style="width:212px;">Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dorsomorphin</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">P5499</td>
			<td style="width:212px;">Compound C; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Embedding medium</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A9011, 0100</td>
			<td style="width:212px;">Mowiol; embedding medium; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Engelbreth-Holm-Swarm (EHS) matrix</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">354277</td>
			<td style="width:212px;">Matrigel hESC-qualified matrix; important: hESC qualified</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fish gelatin&#160;</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">G7765-250ML</td>
			<td style="width:212px;">Gelatin from cold water fish skin; multiple suppliers; autoclave after adding to PBS to dissolve and sterilize, store at 4&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glycine</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A1067,1000</td>
			<td style="width:212px;">Glycine for molecular biology; multiple suppliers&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inoculation loop with needle, disposable (1 &#181;l)</td>
			<td style="width:123px;">Sigma Aldrich, US</td>
			<td style="width:119px;">BR452201-1000EA</td>
			<td style="width:212px;">multiple suppliers&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">I3536-100MG</td>
			<td style="width:212px;">multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-glutamine</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">25030081</td>
			<td style="width:212px;">L-glutamine (200 mM)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium A</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;">#05850</td>
			<td style="width:212px;">mTeSR1 (hiPSC medium)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium B</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;">#05835</td>
			<td style="width:212px;">Neural induction medium (NIM); neural differentiation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medium C</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">21103049</td>
			<td style="width:212px;">Neural Basal Medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">11140035</td>
			<td style="width:212px;">MEM non-essential amino acids solution (100x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MycoAlert Mycoplasma Detection Kit</td>
			<td style="width:123px;">Lonza, Switzerland</td>
			<td style="width:119px;">#LT07-218</td>
			<td style="width:212px;">Mycoplasma detection kit; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nestin</td>
			<td style="width:123px;">Novus biologicals</td>
			<td style="width:119px;">NBP1-92717</td>
			<td style="width:212px;">Nestin mouse monoclonal antibody (4D11)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Paraformaldehyde (PFA)</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A3813, 0500</td>
			<td style="width:212px;">4% in PBS, store solution at -20&#176;C; caution: wear skin and eye protection and work under hood&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS tablets</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">18912014</td>
			<td style="width:212px;">See manufacturer&#180;s instructions; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10.000 U/ml)</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">15140122</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly-L-lysine solution (PLL)</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">P8920-100ML</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pVim</td>
			<td style="width:123px;">MBL</td>
			<td style="width:119px;">D076-3S</td>
			<td style="width:212px;">Phospho-Vimentin (Ser55) mAb</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Reagent A&#160;</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;"># 05872</td>
			<td style="width:212px;">Note to Protocol 1.1.1.2; ReLSR (Enzyme-free human ES and iPS cell selection and passaging reagent); please follow manufactorer&#180;s protocol; alternative products from muliple suppliers available</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Reagent B&#160;</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">A6964-100ML</td>
			<td style="width:212px;">Accutase solution is an enzymatic solution for single cell dissociation; multiple suppliers; protocol 1.1.2 &#34;enzymatic cell dissociation solution&#8221;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Research Cryostat Leica CM3050 S</td>
			<td style="width:123px;">Leica biosystems</td>
			<td style="width:119px;">CM3050 S</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SB431542</td>
			<td style="width:123px;">Selleckchem.com</td>
			<td style="width:119px;">S1067</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spinner flask 250 ml</td>
			<td style="width:123px;">IBS Integra Bioscience</td>
			<td style="width:119px;">182026</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A4734, 1000</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Superfrost ultra plus microscope slides</td>
			<td style="width:123px;">Thermo scientific, US</td>
			<td style="width:119px;">J3800AMNZ</td>
			<td style="width:212px;">Slides should be labeled with a &#34;+&#34; and positively charged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Supplement 1</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">17502048</td>
			<td style="width:212px;">N-2 supplement (100x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Supplement 2 w/o Vitamin A</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">12587010</td>
			<td style="width:212px;">B-27 supplement (50x), minus vitamin A; multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-Tek Cryomold</td>
			<td style="width:123px;">Sakura, NL</td>
			<td style="width:119px;">4565</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:123px;">Sakura, NL</td>
			<td style="width:119px;">4583</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A1388,0500</td>
			<td style="width:212px;">Multiple suppliers multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TUJ1</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">T2200</td>
			<td style="width:212px;">&#946;-Tubulin III (rabbit polyclonal)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TUNEL assay</td>
			<td style="width:123px;">Promega, US</td>
			<td style="width:119px;">G3250</td>
			<td style="width:212px;">DeadEnd Fluorometric TUNEL system; multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20 for molecular biology</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A4974,0500</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">waterproof sheet</td>
			<td style="width:123px;">BEMIS company, inc.</td>
			<td style="width:119px;">PM996</td>
			<td style="width:212px;">Parafilm &#8220;M&#8221;; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Y-27632&#160;</td>
			<td style="width:123px;">Selleckchem.com</td>
			<td style="width:119px;">S1049</td>
			<td style="width:212px;">ROCK-inhibitor (Y-27632 2HCL); multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#946;-mercaptoethanol</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">31350010</td>
			<td style="width:212px;">2-mercaptoethanol (50 mM); multiple suppliers</td>
		</tr>
	</tbody>
</table>

generation of ipsc-derived human brain organoids to model early neurodevelopmental disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

A geração de Organóides cerebrais requer pelo menos três semanas de cultura contínua (Figura 1A). Para conseguir resultados reproduzíveis, recomendamos que o pesquisador documenta cada passo e, sobretudo, evita quaisquer alterações em relação componentes do meio, pontos de tempo e manipulação de célula. Aqui, damos um resumo de como avaliar se marcos críticos são atingidos a fim de obter Organóides de qualidade suficiente no final do experimento. A forma?...

Watch this Scientific Journal Video about Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders at JoVE.com

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

Geração de iPSC-derivado do cérebro Organóides Humanos para modelar alterações precoces do desenvolvimento neurológico

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

generation-ipsc-derived-human-brain-organoids-to-model-early

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

19.6K visualizações.  University of Cologne. Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described. 

Vídeo: Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Detecção rápida de desenvolvimento neurológico fenótipos nas células precursoras neurais humanas (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Biologia do Desenvolvimento

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Geração de padronizada e reprodutível Forebrain-tipo Organoids Cerebral de humanos induzida por células-tronco pluripotentes

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Infecção por vírus Zika do cérebro Fetal humano culta células-tronco neurais para análise de Immunocytochemical

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

Definidos Xeno-free e livre de alimentador condições de cultura para a geração de humanos iPSC-derivado da retina célula modelos

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Modelo in vitro 3D e pipeline computacional para quantificar o potencial Vasculogênico de progenitores endoteliais derivados de iPSC

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Geração de Organoids de vias biliares extra-hepáticas Mouse

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

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 ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

Geração de organóides endometriais primários humanos multicelulares

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Geração de Organóides Mammary mosaicos por Trypsinização Diferencial

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Geração de um chip de barreira hematoencefálica baseada no iPSC humano

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...