This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium &#8220;Tolerage&#8221;, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-W&#252;rttemberg.

Este estudo foi aprovado pelo comitê de ética do Regierungspräsidium Karlsruhe. Todos os animais foram alojados em condições específicas isentos de agentes patogénicos no centro de pesquisa alemão do cancro (DKFZ). Para todas as experiências de cultura de filhotes de rato que variam de 1 a 7 dias de idade foram usados. 1. Isolamento de células mTEC de Thymus NOTA: As etapas seguintes de digestão foram realizadas conforme descrito anteriormente 1 sob condições estéreis, com algumas modificações como se segue. <ol><li> Decapitar os filhotes de rato e remover o timo. Coloque a timos em gelo em uma placa de Petri contendo meio RPMI 1640 (contendo FCS a 5%). </li><li> Corte o thymi em finos, pequenos pedaços e coloque em um tubo de fundo redondo com ~ 5-30 ml de meio RPMI e misture delicadamente usando um ímã para 10 min a RT. </li><li> Depois disso, decantar o sobrenadante contendo principalmente timócitos e digerir o tecido restante sequencialmente com uma rodada de collagenaSE tipo IV (0,2 mg / ml e 57 U / ml de concentartion final) durante 15 minutos cada, a 37 ° C, seguido de colagenase / dispase (0,2 mg / ml e a concentração final de 1,2 U / ml) durante 25 min cada a 37 ° C em um banho de água com agitação magnética até a timos são completamente digerido. Use 1 ml de enzima por 2-3 thymi. </li><li> Agita-se o tecido uma vez a cada 7-10 min com uma pipeta de Pasteur. Reunir as fracções de colagenase / dispase e filtrar através de uma gaze 70 mm. </li><li> Enriqueça as células mTEC por célula magnética triagem (MACS). Realizar a purificação de células mTEC por célula magnética triagem como descrito anteriormente 11, e mostrado na Figura 1. NOTA: Para triagem magnética de células mTEC foram utilizados os seguintes anticorpos: anti-CD80-PE (16-10A1, use a diluição 1: 100) e anti-EpCAM-bio (G8.8, uso a diluição 1: 100) 12. Células mTEC imaturos e maduros usando MACS foram definidos como: CD45 - EpCAM + CD80 - e CD45 - CD80 + respectivEly. </li><li> Após purificação MACS (pureza de células mTEC imaturos = 83,1 ± 6,3% e células mTEC maduros = 79,23 ± 3,42%), semear as células mTEC para as culturas organotípicas conforme descrito abaixo (Seção 2.3). </li><li> Alternativamente, células mTEC ordenar por FACS (utilizando 100 bocal mm) após a depleção de CD45 MACS utilizando os seguintes anticorpos: anti-CD45-PerCP (30-F11, em utilização diluição 1: 100), anti-FITC-Ly51 (6C3, usar a 1 : 100 de diluição), anti-EpCAM-Alexa647 (G8.8, a utilização de 1: 500 de diluição) e utilização de anti-CD80-PE (16-10A1, a diluição 1: 100). Excluir células mortas, utilizando iodeto de propídio (1: 5000) (Dia 4). Células mTEC imaturos e maduros usando FACS foram definidos como: PI - CD45 - Ly51 - EpCAM + CD80 - e PI - CD45 - Ly51 - EpCAM + CD80 +, respectivamente. </li></ol> 2. organotï¿½icas 3D Co-culturas (OTCs) NOTA: As construções em 3D-dérmica para o cultivo de organotípico keratinocytes foram preparados como descrito anteriormente 9,13. Em todos os passos células foram incubadas a 37 ° C e 5% de CO 2. Os OTC utilizando células mTEC foram preparadas com pequenas modificações como se segue. <ol><li> Preparação de fibroblastos humanos NOTA: Os fibroblastos dérmicos humanos foram obtidos a partir de culturas de explantes de derme epidermised-de 9, como descrito anteriormente. <ol><li> Em breve, cortar tiras de pele humana (aproximadamente 5 cm de comprimento) e trata-se com termolisina (0,5 mg / ml em solução salina com 10 mM de Hepes pH 7,4) O / N a 4 ° C. </li><li> Em seguida, separar a epiderme da derme utilizando pinças. </li><li> Finamente cortada a derme em pedaços pequenos, coloque em uma placa de 10 centímetros de Petri e deixar secar durante 1-2 horas sob um fluxo de ar estéril. Suplemento os explantes regularmente com DMEM contendo 20% de FBS. </li><li> Divida os fibroblastos em crescimento fora quando confluentes (geralmente em torno de 3 semanas), utilizando 0,1% de tripsina certificando-se que os explantes continuar a aderir à placa para obter mais conrounds consecutivos de crescimento. </li><li> Expandir os fibroblastos a partir de explantes para os mesmos até 3 vezes em DMEM com FBS a 10% e crio-conservação 14. Utilizar o mesmo lote de fibroblastos para cada série experimental. NOTA: Os fibroblastos não foram irradiadas para o set-up dos equivalentes dérmicos. </li></ol></li><li> Preparação do andaime <ol><li> Corte as 0,4-0,6 mm de espessura de viscose, material fibroso não tecido (detalhes do produto no apoio folha de excel) em círculos bem demarcadas usando uma afiada 11 mm de diâmetro de metal perfurador para caber exatamente em 12 inserções bem-filtro. Em seguida, coloque-o na 12- elemento filtrante bem (poliéster poros da membrana capilar, 3 mm de tamanho de poro) como um andaime. Coloque a configuração completa em um filtro estéril de 12 poços. </li><li> Prepara-se o gel de fibrina utilizando uma cola de fibrina kit para a cirurgia que consiste de uma combinação de f ibrinogénio e trombina. Pré-diluir a fibrinogen-, bem como o componente de trombina do kit para 8 mg / ml e 10unidades / ml, respectivamente. Para um único poço de uma placa de 12 poços proceder da seguinte forma. <ol><li> Dilui-se 100 ul de fibrinogénio (8 mg / ml) com 100 ul de solução salina tamponada de fosfato (PBS) sem Ca 2+ e Mg 2+, pH 7,0. Dilui-se 100 ul de trombina (10 unidades / ml) com 100 ul de FCS contendo 270.000 fibroblastos. </li><li> Dispensar 200 ul de trombina a fibroblastos de células contendo-o para misturar andaime, a qual Adicionar 200 ul de fibrinogénio (mistura 1: 1), resultando numa concentração final de fibrinogénio de 2 mg / ml de trombina e de 2,5 unidades / ml. Misturar bem e distribua uniformemente sobre toda a área do andaime por pipetagem suave (Dia 1). NOTA: Depois de 30 min a 37 ° C de um coágulo que encerra os fibroblastos se ter formado, enchendo completamente os espaços internos do andaime e formar uma superfície superior lisa. </li></ol></li></ol></li><li> Co-cultura com células mTEC <ol><li> Para pré-cultura, mergulhe as culturas de órgãos emDMEM com FBS a 10%, 50 ug / ml de ácido L-ascórbico e de 1 ng / ml de TGF-β1 com uma mudança de meio todos os outros dias durante 4-5 dias. </li><li> No dia da sementeira MTEC, substituir o meio por meio RFAD (1: 1 DMEM + DMEM / F12) com 10% de FBS, 10 -10 M a toxina da cólera, 0,4 mg / ml de hidrocortisona, 50 ng / ml de ácido L-ascórbico, ligando RANK (0,1 ug / ml) e 500 unidades / ml de aprotinina, evitando assim a fibrinólise precoce por serina-proteases secretadas pelos fibroblastos. </li><li> 7-8 horas mais tarde, as set-up co-culturas, semeando 250.000 células mTEC (quer células mTEC completos, ou CD80 lo e subconjuntos CD80 hi), num volume de 100 ul por poço em cima do andaime de fibroblastos. Contar as células usando uma câmara de Neubauer (dia 4). NOTA: Em todas as vezes as culturas organotípicas 3D timo estão submersos em meio ao contrário OTCs pele que são levantadas ao ar. </li><li> Após 24 horas de incubação, fornecer as culturas com meio (câmbio médio total) como mencionado acima (passo 2.3.2) agora containing reduziram quantidades de aprotinina, 250 unidades / ml (dia 5). </li><li> A fim de avaliar a actividade proliferativa de células mTEC, adicionar EdU (6,7 uM / ml, ou seja, 10 pM / poço) para OTC durante 4 horas antes da terminação das culturas. Realizar a coloração de OTC crio-secções, como descrito no kit de EdU de imagem conjugados com co-coloração de queratina 14. Determinar os índices proliferativas de células mTEC, tanto por contagem de células K14 + edu + de duas secções de cada espécime de cultura ou por fluxo citometria (EdU citometria de fluxo, Secção 1.7, mas em vez de utilizar Ly51-FITC CDR1 PB-15 a uma diluição de 1: 100 e 2.3.6.4). </li><li> Na sequência de 4-7 dias de co-cultura, encerrar os OTCs e processo para isolamento do RNA, crio-seccionamento ou análise FACS (dia 11/08). <ol><li> Terminar as culturas usando uma pinça, que separa o andaime / equivalente dérmico do filtro do poço de inserção. </li><li> Para crio-seccionamento, incorporar toda a OTC em composto outubro e congelar na livapor de nitrogênio quid antes de crio-seccionamento. Prepare 5-7 uM secções espessas OTC utilizando um criostato e armazenar a -20 ° C até à sua utilização. Para imuno-histoquímica de OTC crio-seccionado usar anti-queratina 14 (AF64, a utilização de 1: 1000 de diluição), e anti-vimentina (gp53, utilização em diluição 1: 100) de anticorpos. Realizar a coloração imuno-histoquímica indireta usando anticorpos secundários respectivos. </li><li> Para o isolamento do RNA, adicionar 1 ml de solução (contendo fenol e tiocianato de guanidina) em uma tampa de rosca de um 2 ml RNase livre tubo de desnaturação. Cortar toda a OTC em pedaços com um bisturi e adicionar para dentro do tubo contendo solução de desnaturação. Mecanicamente triturar os OTC com instrumento FastPrep duas vezes durante 30 segundos a uma velocidade de 6,0, em colocar entre em gelo durante 2 minutos (a amostra pode ser armazenada a -80 ° C após este ponto). Se congeladas a -80 ° C, descongelar em gelo. Centrifugar os tubos a 11,500-13,000 rpm durante 10 min a 4 ° C. Transferir o sobrenadante para um tubo fresco, incubar durante 5 min à temperatura ambiente e fprotocolos de isolamento de ARN Realize as utilizando ácido extracção tiocianato de guanidina-fenol-clorofórmio, tal como descrito pelo fabricante. </li><li> Para a análise FACS, remover o andaime e separar a membrana da fibrina / fibroblastos / gel MTEC. Finamente cortada do gel com um bisturi e digeri-la num tubo de FACS durante ~ 20 min, ou até estarem completamente digeridas com 2 ml de colagenase / dispase a 37 ° C num banho de água com agitação magnética. Agita-se a solução de enzima com uma pipeta de Pasteur, uma vez a cada 5 minutos. Após a digestão completa, filtra-se a suspensão de células através de um filtro de 70 um; manchar a suspensão de célula única usando os anticorpos, tal como descrito em 1.7 e análise por citometria de fluxo. </li></ol></li></ol></li></ol>

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Os autores declaram que não há conflito financeiro ou comercial de interesse.

Ao lado RTOCs, o OTCs 3D têm sido, de longe, superior em termos de diferenciação do TCE e PGE manutenção / indução (Tabela 1) em comparação com outros (i) &#39;culturas 3D simplificadas&#39; usando - fibroblastos sozinho, sem o cadafalso; (Ii) sistemas 2D utilizando - células fibroblastos / alimentador de co-cultivadas com TEC 10, (iii) células 3T3-J2 em que os clones TEC desenvolver, mas é perdida PGE, (iv) Matrigel ou (v) de componentes da matriz extracelular (dados não publica...

Timócitos desenvolvimento perfazem cerca de 98% do timo, enquanto que os restantes 2% é composto por uma variedade de células que compõem colectivamente o estroma tímico (isto é, células epiteliais, células dendríticas, macrófagos, células B, f ibroblastos, células endoteliais). As células epiteliais cortical exterior (CTECs) adquirir imigração de células pro-T a partir de medula óssea, t indução linhagem celular em células pré-T multipotentes e selecção positivo de auto-MHC restrita timócitos imaturos. As células interiores medulares do timo epiteliais (células mTEC) estão envolvidas na indução de tolerância desses timócitos com um TCR de elevada afinidade para complexos de auto-péptido / CPH por indução ou selecção negativa ou seu desvio para a linhagem de células T reguladora. No contexto da indução de tolerância central, células mTEC são únicos na medida em que expressam uma ampla gama de auto-antigénios restrita a um tecido (TRAs) espelhamento assim a auto periférica. Este fenômeno é chamado de expressão gênica promíscua (PGE)1,2. A maioria dos estudos actuais sobre este tipo de célula fascinante dependem ex vivo células isoladas, como vários sistemas de cultura de curto prazo 2D invariavelmente resultou na perda de moléculas de PGE e como regulador chave do MHC de classe II, e Foxn1 Aire dentro dos primeiros 2 dias 06/03 . Ele permaneceu no entanto claro, que determinados componentes e características da malha 3D intacta do timo estavam faltando em modelos 2D. A cultura de órgão tímico re-agregação (RTOC) tem sido até agora o único sistema 3D, que permite o estudo do desenvolvimento de células T, por um lado, e da biologia celular de estroma, por outro lado, em um micro-ambiente do timo 7 intacto. No entanto, RTOCs tem certas limitações, isto é, que já contêm uma mistura complexa de células, requerem a utilização de células estromais fetais e suportar um período de cultura máxima de 5 a 10 dias. A falta de redutora em sistemas de cultura in vitro tem dificultado o estudo dovários aspectos do desenvolvimento de células T do timo e organogênese não menos importante a regulação molecular da PGE e sua relação com a biologia do desenvolvimento de células mTEC. Devido à estreita-relação da organização estruturada das células epiteliais da pele e do timo, optamos por um sistema 3D cultura organotípicas (OTC), que tinha sido desenvolvido originalmente para emular a diferenciação de queratinócitos in vitro e, assim, criar um equivalente dérmico. O sistema OTC consiste em uma matriz de andaime inerte coberto com fibroblastos dérmicos que estão presos em um gel de fibrina, na qual os queratinócitos são semeados 8,9. Aqui, nós substituímos queratinócitos com células mTEC purificados. Mantendo as características básicas deste modelo, nós otimizamos determinados parâmetros. No modelo adotado OTC células mTEC proliferaram, foram submetidos a diferenciação terminal e mantido identidade MTEC e PGE, assim estreitamente imitando in vivo células mTEC desenvolvimento <sup&gt; 10. Esta nota técnica proporciona um protocolo detalhado permitindo que o passo a passo set-up de OTCs timo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 mice&#160;</td>
			<td>Charles River WIGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Microbeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (G8.8 -Alexa 647 and -biotin)</td>
			<td>Ref. 12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD80-PE antibody</td>
			<td>BD Pharmingen</td>
			<td>553769</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-PerCP antibody</td>
			<td>BD Pharmingen</td>
			<td>557235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly51-FITC antibody</td>
			<td>BD Pharmingen</td>
			<td>553160</td>
			<td></td>
		</tr>
		<tr>
			<td>CDR1-Pacific Blue</td>
			<td>Ref. 15</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 14 antibody</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin antibody</td>
			<td>Progen</td>
			<td>GP58</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated AffiniPure F(ab&#39;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>106-546-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Molecular Probes (Invitrogen GmbH)</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">Click-iT EdU Alexa Fluor 594 Imaging Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">C10339</a></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">C10425</a></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well filter inserts (thincerts)</td>
			<td>Greiner bio-one</td>
			<td>657631</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Greiner</td>
			<td>665180-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Jettex 2005/45</td>
			<td>ORSA, Giorla Minore, Italy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Serva</td>
			<td>47302.03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Lonza</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-049&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Gold</td>
			<td>GE Healthcare</td>
			<td>A11-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin (Trasylol)</td>
			<td>Bayer</td>
			<td>4032037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Biomol</td>
			<td>G117</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Seromed (Biochrom)</td>
			<td>K3520</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#223;1</td>
			<td>Invitrogen</td>
			<td>PHG9214</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>R&#38;D systems</td>
			<td>462-TR-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysin</td>
			<td>Sigma Aldrich</td>
			<td>&#160;T-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>TissueTek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (aka. Denaturing solution - Acid guanidinium thiocyanate-phenol-chloroform extraction)</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		</tr>
		<tr>
			<td>FastPrep FP120</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV&#160;</td>
			<td>CellSystems</td>
			<td>LS004189</td>
			<td>0.2 mg/ml and 57U/ml final conc.</td>
		</tr>
		<tr>
			<td>Neutrale Protease (Dispase)</td>
			<td>CellSystems</td>
			<td>LS002104</td>
			<td>0.2 mg/ml and 1.2U/ml final conc.</td>
		</tr>
		<tr>
			<td>DNase I&#160;</td>
			<td>Roche</td>
			<td>11 284 932 001</td>
			<td>25 &#181;g/ml final conc.</td>
		</tr>
	</tbody>
</table>

3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression

Desenvolvimento de células T intra-timo requer uma malha tridimensional complexa composta por várias células do estroma, ou seja, células não-T. Timócitos atravessar este andaime em uma ordem temporal e espacial altamente coordenada enquanto sequencialmente passar pontos de verificação obrigatórios, ou seja, T compromisso linhagem de células, seguido de geração repertório receptor de células T e seleção antes da sua exportação para a periferia. Os dois tipos de células residentes principais que formam este andaime são células epiteliais do timo corticais (CTECs) e medular (células mTEC). Uma característica chave de células mTEC é o chamado promíscuo expressão de numerosos antigénios restrita a um tecido. Estes antigénios restrita a um tecido são apresentados para os timócitos imaturos, directa ou indirectamente por células mTEC ou células dendríticas do timo, respectivamente, resultando em auto-tolerância. Adequados in vitro modelos emulando as vias de desenvolvimento e as funções de CTECs e células mTEC Atualmente lacrei. Esta falta de modelos experimentais adequados, por exemplo, tem dificultado a análise da expressão do gene promíscuo, que ainda está mal compreendida no nível celular e molecular. Nós adaptamos um modelo de co-cultura 3D organotípico à cultura ex vivo células mTEC isoladas. Este modelo foi inicialmente criado para cultivar queratinócitos, de modo a gerar um equivalente de pele in vitro. O modelo 3D preservou as características funcionais do MTEC biologia: (i) a proliferação e diferenciação terminal de CD80 lo, Aire-negativo em CD80 oi, Aire-positivo células mTEC, (ii) capacidade de resposta a RANKL, e (iii) a expressão sustentada de Foxn1, Aire e genes restrita de tecidos em células mTEC CD80 hi.

Adotamos um modelo 3D organotípico co-cultura (3D OTC) que tinha sido originalmente desenvolvido para o cultivo in vitro de longa duração dos queratinócitos 9. Células mTEC MACS enriquecidos (ver esquema de enriquecimento MACS Figura 1) foram semeadas em um andaime que compreende de um gel de fibrina e fibroblastos aprisionadas. Os fibroblastos proporcionar a matriz extracelular essencial (ECM) que suporta células mTEC in vitro. Células mTEC foram cultivadas ...

Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D Organotípicas Co-cultura Modelo de Apoio medular Thymic Epithelial proliferação, diferenciação e Expression Promiscuous Gene

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

3d-organotypic-co-culture-model-supporting-medullary-thymic

Research

JoVE Journal

Developmental Biology

10.6K visualizações.  German Cancer Research Center (DKFZ). Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression. 

Vídeo: 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Cultura e Co-Culture of Mouse ovários e folículos ovarianos

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Biologia do Desenvolvimento

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Previsão e Validação de elementos reguladores de genes ativada durante ácido retinóico induzida estaminais embrionárias Diferenciação Celular

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Adenoviral terapia genética para Diabetic Ceratopatia: Efeitos sobre a cicatrização de feridas e Stem Cell marcador expressão em humanos Corneas Organ-cultivadas e células epiteliais Limbal

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Ordenação temporal de Expressão Dynamic Data de expressão espacial mapas detalhados

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

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

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

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

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

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

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

Protocolo de diferenciação cerebelar 3D simplificada com modificação 2D opcional

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

Análise clonal de precursores embrionárias células-tronco hematopoiéticas usando índice de célula única classificação combinada com células endoteliais nicho co-cultura

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Proliferação e diferenciação de murino Precursor mieloide 32G/G-CSF-R células

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

Reprogramação hemogénica de fibroblastos humanos pela expressão forçada de fatores de transcrição

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

Um modelo de expressão de Quimiocina ectópica para testar o recrutamento de macrófago in vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...