Os autores gostaria de agradecer os Drs Stephanie Spohn e Basileia Abuaita para muitas discussões úteis na microinjeção de organoides. JRS é suportado pelo Intestinal células estaminais Consortium (U01DK103141), um projeto colaborativo de pesquisa financiado pelo Instituto Nacional de Diabetes e digestivo e doenças do rim (NIDDK) e o Instituto Nacional de alergia e doenças infecciosas (NIAID). JRS e VBY são suportados pelo romance NIAID, sistemas de modelo alternativo para o consórcio de doenças entéricas (NAMSED) (U19AI116482). DRH tem suporte a mecanismos de patogénese microbiana formação concessão do Instituto Nacional de alergia e doenças infecciosas (NIAID, T32AI007528) e o prêmio de ciência translacional e clínica ao Instituto Michigan para clínica e saúde Pesquisa (UL1TR000433).
Os arquivos de dados completos e o código de análise de dados usado neste manuscrito estão disponíveis em https://github.com/hilldr/HIO_microinjection.

<ol>
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Os autores têm sem concorrentes interesses financeiros ou outros conflitos de interesse a divulgar.

Este protocolo estabelece um método de uso geral para a microinjeção de HIOs hPSC-derivados e derivados de tecido intestinal organoids e a medição da permeabilidade da barreira epitelial em tempo real. Também demonstrámos a nossa abordagem para análise e interpretação dos dados gerados usando estes métodos. Dada a crescente adoção do organoids intestinal sistemas modelo16,20,21,28 e o interesse de longa data na permeabilidade da barreira intestinal como um fisiologicamente relevantes funcional resultado3,4,5,6, prevemos que outras pessoas que trabalham neste campo será capazes de aplicar e construir sobre esses métodos.
Há várias etapas que são essenciais para a aplicação desta técnica. Acesso ao hPSC de alta qualidade - ou tecido derivada HIO de tecido deve ser estabelecido antes da experimentação extensiva com microinjeção. HIO macrostructure pode ser heterogêneo, com variação no tamanho e na forma, embora a identidade de tecido e a morfologia celular é altamente reprodutível quando utilizando a metodologia estabelecida para gerar HIOs24. HIOs esféricas, consistindo de um único lúmen semi-transparente e medindo aproximadamente 1 mm de diâmetro são ideais para microinjeção e medição de fluorescência luminal em tempo real. Em alguns casos, microinjeção irá falhar, resultando em colapso do HIO ou óbvio vazamento de material injetado. HIOs falhadas podem ser removidos da cultura bem a critério do usuário usando uma micropipeta padrão. Considere as lentes objetivas disponíveis em uma plataforma de imagem ao selecionar HIOs para microinjeção e imagem. Em geral, 2-4 X objectivas são ideais para capturar o sinal fluorescente completo do HIO, embora um objetivo X 10 pode ser utilizado se as lentes de baixa potência não estiverem disponíveis ou se o HIOs disponíveis são &#60; 1 mm de diâmetro. Software de imagem deve permitir a captura automatizada de imagens fluorescentes em pontos definidos ao longo do tempo.
Diversas modificações do presente protocolo são possíveis a fim de atender às necessidades de experimentais. Por exemplo, os resultados dos testes de função de barreira podem ser dependentes do tamanho molecular dos compostos em uso43 e pode ser apropriado testar preparações de dextrano de variação de peso molecular. Além disso, pode efectuar-se a imagem brightfield além de imagens de fluorescência como um indicador da integridade estrutural global do tecido25. Ao executar a microinjeção de bactérias vivas25,28,31,32,33,44, pode ser necessário adicionar penicilina e estreptomicina ou gentamicina para os meios de cultura HIO antes ou depois da microinjeção. Fora da conta vai ser contaminada durante o enchimento com a suspensão da cultura bacteriana e isto poderá ser transferido para a mídia HIO. Alternadamente, microinjeção pode ser executada em HIOs suspendidos na matriz extracelular (por exemplo, Matrigel) sem mídia, adicionando a mídia após a conclusão da microinjeção. Isto pode limitar a contaminação para a matriz extracelular e a face externa do HIO. Ao planejar a ensaios de crescimento microbiano, pode ser necessário remover os antibióticos na mídia após 1-2 h para evitar, retardar ou impedir o crescimento de organismos microinjected.
Finalmente, reconhecendo que nem todos os pesquisadores terão acesso a equipamentos de microscopia, adequado para a imagem latente em vitro , é importante salientar que os procedimentos descritos no presente protocolo para coleta de dados de fluorescência podem ser aplicados a imagens tomadas em momentos fixos usando microscopia epifluorescente padrão sem captura automatizada de imagem ou controles ambientais. Exemplos desta abordagem podem ser encontrados nos relatórios por Leslie e Huang et al . 25, que examinaram a atividade de toxina de c. difficile no hPSC-derivado organoids intestinal e Karve e Pradan et al 44, que examinaram a permeabilidade da barreira epitelial em semelhante organoids intestinal hPSC-derivado, injetadas ao vivo e. coli. Operação manual do equipamento de imagem pode resultar em maior variação e dificuldade na normalização do sinal fluorescente. Quando executar imagem manual do FITC-dextran injetado HIOs é essencial para manter a ampliação fixa, intensidade de excitação fluorescente e tempos de exposição em todo o experimento para evitar distorcer as medições de intensidade fluorescente.

O epitélio intestinal forma uma barreira seletiva que medeia o direcional transporte de nutrientes, H2O, íons e resíduos de produtos, minimizando inespecífica troca mediada por difusão de outras partículas entre o lúmen e o mesenquimais tecido ou sangue fornecimento1,2. A permeabilidade inespecífica da barreira epitelial intestinal tem sido considerada um parâmetro chave funcional na saúde e na doença,3,4,5,6, que reflete a taxa de difusão de pequenas moléculas através do epitélio através do espaço paracellular. Medição da permeabilidade da barreira epitelial pode efectuar-se em animal modelos7 e em pacientes humanos8 através da ingestão de lactulose, que não tem nenhum transportador específico no tracto gastrointestinal e a coleção subsequente e a medição das concentrações de lactulose no sangue periférico. Marcadores ingeridos alternativos da função de barreira, tais como hidratos de carbono fluorescente etiquetadas também estão disponíveis9,10. Esta abordagem foi adaptada para a célula epitelial intestinal culturas cultivadas em Transwell suporta11, uma abordagem simplificada que permite maior controle experimental, mas também tem sido criticada como um preditor pobre da vivo em permeabilidade devido à ausência de subtipos epiteliais diferenciados e estrutura de tecido12. Utilizando câmaras representam ainda uma outra abordagem e permitir a medição da função de barreira epitelial na mucosa intestinal todo ex vivo13. Aplicação desta técnica é frequentemente limitada pelo tecido disponibilidade e condição13,14. Assim, novos métodos que equilibrem a reprodutibilidade e a taxa de transferência com relevância fisiológica são necessários.
Desenvolvimentos recentes na organogênese em vitro conduziram à adopção de sistemas de cultura de tecido 3D modelo como uma plataforma sofisticada para recapitulando a dinâmica de tecidos complexos15,16,17 ,18,19,20,21,22,23. Em particular, células-tronco (hPSC) derivada de pluripotentes humanas organoids intestinal humana (HIOs)19,24 surgiram como um sistema reprodutível e experimentalmente tractable modelo para o estudo das interações hospedeiro-microbiana e barreira epitelial dinâmica25,26,,27,28. Da mesma forma, organoids de derivado de tecido humano (também conhecido como enteroids) pode ser derivado de um procedimento de biópsia simples e pode ser usado como um sistema tractable para estudar a fisiologia humana e doença15,29,30. Microinjeção de organoids intestinal humano permite a entrega de compostos experimentais25 ou vive micróbios25,31,32,33 para a apical epiteliais superfície do lúmen de organoides. Leslie e Huang et al . 25 recentemente adaptado a esta técnica para medir a permeabilidade de barreira em HIOs injetadas com isotiocianato de fluoresceína (FITC) rotulado dextrano após a exposição a toxinas bacterianas.
Este protocolo destina-se como um guia para a medição da permeabilidade da barreira epitelial em HIOs hPSC-derivados e derivados de tecido HIOs usando microscopia fluorescente. Com pequenas modificações, ele também pode servir como um primer geral na microinjeção de HIOs com compostos experimentais. Os usuários podem complementar este protocolo com aplicações a jusante adicionais ou alternativos após microinjeção.

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	<tbody>
		<tr>
			<td>EGTA 0.5 M sterile (pH 8.0)</td>
			<td>Bioworld</td>
			<td>405200081</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell matrix solution (Matrigel)</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Deltavision RT live cell imaging system</td>
			<td>GE Life Sciences</td>
			<td>29065728</td>
			<td>http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/brands/deltavision/</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>GE Life Sciences</td>
			<td>29065728</td>
			<td>Included with Deltavision system</td>
		</tr>
		<tr>
			<td>softWoRx Imaging software</td>
			<td>GE Life Sciences</td>
			<td>29065728</td>
			<td>Included with Deltavision system</td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Labconco</td>
			<td>Cell Logic+</td>
			<td>http://www.labconco.com/product/purifier-cell-logic-class-ii-type-a2-biosafety-cabinets-2/4262</td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM-F12</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>B27 supplement (50X)</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>L-glutamine (100X)</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>Manipulator</td>
			<td>Narshge</td>
			<td>UM-3C</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narshge</td>
			<td>UM-1PF</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Holder</td>
			<td>Narshge</td>
			<td>UP-1</td>
			<td>Alternate to Xenoworks pipette holder</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Narshge</td>
			<td>GJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scope</td>
			<td>Olympus</td>
			<td>SX61</td>
			<td>Recommended scope, although other models are likely compatible</td>
		</tr>
		<tr>
			<td>Olympus IX71 Fluorescent microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td>Included with Deltavision system</td>
		</tr>
		<tr>
			<td>CoolSNAP HQ2</td>
			<td>Photometrics</td>
			<td>29065728</td>
			<td>Included with Deltavision system</td>
		</tr>
		<tr>
			<td>Recombinant C. difficile Toxin A/TcdA Protein</td>
			<td>R&#38;D Systems</td>
			<td>8619-GT-020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;D Systems</td>
			<td>236-EG</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>R-spondin 1</td>
			<td>R&#38;D Systems</td>
			<td>4645-RS</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>R&#38;D Systems</td>
			<td>6057-NG</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-dextran (4 kDa)</td>
			<td>Sigma-Aldrich</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek II Chamber Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>154526PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass filaments</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Xenoworks</td>
			<td>BR-MH2</td>
			<td>Preferred device</td>
		</tr>
		<tr>
			<td>Analog Tubing kit</td>
			<td>Xenoworks</td>
			<td>BR-AT</td>
			<td></td>
		</tr>
		<tr>
			<td>1/16 in clear ferrule</td>
			<td>Xenoworks</td>
			<td>V001104</td>
			<td></td>
		</tr>
		<tr>
			<td>1-1.2 mm O-ring</td>
			<td>Xenoworks</td>
			<td>V300450</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time measurement of epithelial barrier permeability in human intestinal organoids

Avanços na cultura 3D dos tecidos intestinais, obtidos através de biópsia ou gerados a partir de células-tronco pluripotentes através de diferenciação direcionada, resultaram em modelos sofisticados em vitro da mucosa intestinal. Aproveitar estes sistemas emergentes do modelo exigirá adaptação de ferramentas e técnicas desenvolvidas para animais e sistemas de cultura 2D. Aqui, descrevemos uma técnica para medir a permeabilidade da barreira epitelial no organoids intestinal humana em tempo real. Isso é realizado pela microinjeção de dextrano fluorescente-etiquetadas e imagem em um microscópio invertido equipado com filtros de epifluorescente. Medição em tempo real da permeabilidade de barreira no organoids intestinal facilita a geração de dados de alta resolução temporais, no tecido epitelial intestinal humano, embora esta técnica também pode ser aplicada a Commit fixo abordagens de imagem. Este protocolo é facilmente adaptável para a medição da permeabilidade de barreira epitelial após a exposição a agentes farmacológicos, produtos bacterianos ou toxinas ou microorganismos vivos. Com pequenas modificações, esse protocolo também pode servir como um primer geral na microinjeção de organoids intestinal e os usuários podem escolher completar esse protocolo com aplicações a jusante adicionais ou alternativas após microinjeção.

HIOs eram diferenciadas das células-tronco pluripotentes humanas e cultivadas em solução de matriz celular como anteriormente descrito19,24. Após 4 semanas de cultura, os HIOs tinham expandido suficientemente para permitir a microinjeção. HIOs foram injetadas com 4 kDa conjugado FITC dextrano suspenso em PBS ou PBS contendo toxina de Clostridium difficile TcdA. C. difficile é um micróbio patogénico oportunista gastrointestinal que apresenta toxicidade epitelial mediada por toxina em HIOs25. Como um controle positivo, EGTA foi adicionado para os meios de cultura HIO em um subconjunto de HIOs injetado com PBS e FITC-dextrano. EGTA é um quelante de cálcio que provoca rápida de polimerização do citoesqueleto de actina42. Fluorescência FITC foi monitorizada em tempo real em um microscópio de imagens ao vivo dentro de uma câmara de ambiente controlado e imagens foram capturadas em intervalos de 10 minutos.
Análise post-hoc de dados de imagem revelou diferenças substanciais na retenção de fluorescência FITC (Figura 5). HIOs injetados com PBS mantido quase todos do sinal fluorescente presente em t = 0, porém HIOs que também foram injetados com TcdA de tratados com EGTA exibiram um decréscimo substancial na intensidade fluorescente por microinjeção de 8 horas pós (Figura 5a). dados de imagens de foram quantificados para HIOs todos em todos os pontos de tempo gerar um conjunto de dados de alta resolução, que representa a mudança relativa em intensidade fluorescente ao longo do tempo em cada condição experimental (Figura 5B). Diferenças na permeabilidade epitelial avaliaram-se calcular o tempo médio de eliminação (t1/2 ) de FITC para cada grupo de tratamento (tabela 1), e comparando as diferenças em t t1/2 entre grupos usando o Student t-teste. Controle-Tratado HIOs reteve a maioria do sinal fluorescente de FITC por mais de 16 horas (t1/2 = 17 ± 0,3 h). Tratamento com EGTA reduziu significativamente o tempo de eliminação FITC-dextrano relativo controle HIOs (t1/2 = 2,6 ± 0,2 h; P = 1,3 x 10-8). Consistente com os resultados publicados anteriormente25, microinjeção de TcdA aumentou significativamente permeabilidade da barreira epitelial em relação ao tratamento controle (t1/2 = 7,6 ± 0,6 h; P = 5,4 x 10-4). Assim, tanto externo (EGTA) e microinjected (TcdA) compostos são capazes de induzir alterações significativas na permeabilidade da barreira epitelial em HIOs. Estes resultados sugerem que os efeitos de uma vasta gama de agentes farmacológicos, metabólitos, produtos bacterianos, citocinas, fatores de crescimento e outros compostos na função de barreira epitelial podem ser avaliados usando esta abordagem.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tratamento</td>
			<td>Meia-vida (h)</td>
			<td>SEM (h)</td>
			<td>Inferior a 95% CI (h)</td>
			<td>Superior a 95% CI (h)</td>
			<td>n</td>
		</tr>
		<tr>
			<td>Controle</td>
			<td>17,11</td>
			<td>0.3</td>
			<td>16.45</td>
			<td>17.78</td>
			<td>11</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>2,58</td>
			<td>0,19</td>
			<td>1.78</td>
			<td>3.38</td>
			<td>3</td>
		</tr>
		<tr>
			<td>TcdA</td>
			<td>7.56</td>
			<td>0,63</td>
			<td>5.93</td>
			<td>9.18</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
Tabela 1: tempo de eliminação (t1/2) para FITC-dextran em HIOs tratados com EGTA ou TcdA. As unidades são pós-microinjeção de horas.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56960/56960fig1.jpg"> 
Figura 1: layout básico de uma microinjector e micromanipulador para microinjeção de HIO. Esta imagem mostra o complemento total de equipamentos utilizados para a realização de microinjeção de HIOs. Componentes-chave são rotulados e ordenação de informações pode ser encontrado na tabela de materiais. <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56960/56960fig2.jpg"> 
Figura 2: extrator de micropipeta. O cobre, aquecimento da bobina hc, braçadeira superior c1, braçadeira inferior (c2), braço extrator (pa), alavanca de seleção de calor (ht) são identificados pelas setas. A configuração correta para o HEAT 1 e puxe são indicadas no texto em vermelho. A inserção mostra um filamento de vidro corretamente montado pronto para o aquecimento. <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56960/56960fig3.jpg"> 
Figura 3: imagens representativas de um derivado de tecido HIO injetado com FITC-dextrano. Imagem do (A) demonstrando o posicionamento da conta de vidro apenas antes da inserção no HIO e microinjeção. (B) Brightfield imagem de um derivado de tecido humano intestinal organoids depois da microinjeção de FITC-dextrano. Note que o sinal de fluorescência é aparente mesmo sem o uso de um conjunto de filtro específico. Esta coloração auxilia precisão microinjeção. 3 ampliação X <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig3large.jpg" target="_blank">, por favor clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56960/56960fig4.jpg"> 
Figura 4: viver o trabalho de imagem. (A) Screenshot ilustrando as etapas necessárias para definir os parâmetros de microscópio, encontrar os HIOs no slide e salvar a posição da fase para cada HIO ser fotografada. Configurações de pré-imagem (B) para capturar o canal FITC em intervalos regulares em cada uma das posições especificadas na exportação de pós-imagens r. (C) de dados em formato DV arquivos como imagens TIFF com uma imagem TIFF por HIO por ponto de tempo.<a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56960/56960fig5.jpg"> 
Figura 5: resultados representativos. (A)-células tronco derivadas organoids intestinal humana (HIO) injetadas com FITC-dextrano de 2 mg/mL (4 kDa) fotografada por 20 horas. HIOs também foram injetadas com PBS (controle) ou toxina Clostridium difficile TcdA (12,8 ng / µ l) ou tratados com 2mm EGTA adicionado à cultura mídia externa. Ampliação de 4x. (B) parcela de média intensidade FITC normalizada ao longo do tempo em HIOs tratados com PBS (controle), TcdA ou EGTA. Barras de erro representam no MEV mostrou e n = 11 HIOs (controle), 3 HIOs (EGTA), 6 HIOs (TcdA). <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig5large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Este protocolo descreve a medição da permeabilidade da barreira epitelial em tempo real seguir tratamento farmacológico em organoids intestinal humana, usando microscopia fluorescente e vive microscopia de célula.

Medição em tempo real da permeabilidade da barreira epitelial no Organoids Intestinal humana

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

real-time-measurement-epithelial-barrier-permeability-human

Research

JoVE Journal

Developmental Biology

14.0K visualizações.  University of Michigan. Este protocolo descreve a medição da permeabilidade da barreira epitelial em tempo real seguir tratamento farmacológico em organoids intestinal humana, usando microscopia fluorescente e vive microscopia de célula.

Vídeo: Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Kinetic Medição e visualização em tempo real de Reprogramação Somatic

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Biologia do Desenvolvimento

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Imuno-fluorescente rotulagem de microtúbulos e proteínas Centrosomal em tecido Intestinal Ex Vivo e 3D em Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Engenharia genética de organóides intestinais do camundongo primário usando vetores virais de transdução de nanopartículas magnéticas para seccionamento congelado

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

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 ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Imagens de bioluminescência em tempo real da dinâmica de sinalização de notch durante a neurogênese de Murine

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

Recapitulando a transição de amamentação in vitro usando organóides intestinais fetais

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

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 ...

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Imagem em tempo real da migração induzida por CCL5 de células-tronco esqueléticas periósteis em camundongos

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Estabelecendo organoides do dente humano como uma poderosa ferramenta para pesquisa mecanicista e terapia regenerativa

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

Medição em tempo real e repetida do crescimento muscular esquelético em peixes-zebra vivos individuais submetidos a atividade elétrica alterada

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...