Agradecemos K. Deisseroth e S. Je para as construções de lentivírus ChR2 e Synapsin1-tdTomato respectivamente, C. Chai para a partilha de conhecimentos e na geração de protocolo iPSC, WY Leong para o suporte técnico, membros do laboratório Goh para reagentes e experiência de compartilhamento. Este trabalho foi financiado pela Abbott Nutrition e do Centro Acadêmico de Excelência (ACE) prêmio de pesquisa da GlaxoSmithKline (GSK) para ELG, pela Fundação de Pesquisa Nacional de Cingapura no âmbito do seu Programa de Pesquisa Competitiva (NRF 2008 NRF-CRP 002-082) para ELG e GJA, e pelo Instituto de Classe Mundial (WCI) Programa da Fundação Nacional da Coreia Pesquisa (NRF), financiado pelo Ministério da Educação, Ciência e Tecnologia da Coreia (MEST) (NRF Grant Number: WCI 2009-003) para GJA

<ol>
	<li>Wang, H., 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=High-speed+mapping+of+synaptic+connectivity+using+photostimulation+in+Channelrhodopsin-2+transgenic+mice.">High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice.</a> Proceedings of the National Academy of Sciences. 104 (19), 8143-8148 (2007).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale genetically targeted optical control of neural activity.</a> Nature Neuroscience. 8 (9), 1263-1268 (2005).</li><li>Nagel, G., 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proceedings of the National Academy of Sciences. 100 (24), 13940-13945 (2003).</li><li>Zhang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+fast+optical+interrogation+of+neural+circuitry.">Multimodal fast optical interrogation of neural circuitry.</a> Nature. 446 (7136), 633-639 (2007).</li><li>Stroh, A., 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=Tracking+stem+cell+differentiation+in+the+setting+of+automated+optogenetic+stimulation.">Tracking stem cell differentiation in the setting of automated optogenetic stimulation.</a> Stem Cells. 29 (1), 78-88 (2011).</li><li>Weick, J. P., Liu, Y., Zhang, S. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+neurons+adopt+and+regulate+the+activity+of+an+established+neural+network.">Human embryonic stem cell-derived neurons adopt and regulate the activity of an established neural network.</a> Proceedings of the National Academy of Sciences. 108 (50), 20189-20194 (2011).</li><li>Viviani, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+coculture+of+neurons+and+glial+cells.">Preparation and coculture of neurons and glial cells.</a> Curr Protoc Cell Biol. 32, 2.7.1-2.7.2.1 (2006).</li><li>Braun, H., G&#252;nther-Kern, A., Reymann, K., Onteniente, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+differentiation+of+human+iPS-cells+in+a+rat+cortical+primary+culture.">Neuronal differentiation of human iPS-cells in a rat cortical primary culture.</a> Acta Neurobiologiae Experimentalis. 72 (30), 219-229 (2012).</li><li>Shivaraj, M. C., 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=Taurine+induces+proliferation+of+neural+stem+cells+and+synapse+development+in+the+developing+mouse+brain.">Taurine induces proliferation of neural stem cells and synapse development in the developing mouse brain.</a> PLoS ONE. 7, e42935 (2012).</li><li>Okita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nature Methods. 8 (5), 409-412 (2011).</li><li>Li, W., 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=Rapid+induction+and+long-term+self-renewal+of+primitive+neural+precursors+from+human+embryonic+stem+cells+by+small+molecule+inhibitors.">Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors.</a> Proceedings of the National Academy of Sciences. 108 (20), 8299-8304 (2011).</li><li>Chen, G., 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=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nature Methods. 8 (50), 424-429 (2011).</li><li>Packer, A. M., Roska, B., Hausser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+neurons+and+photons+for+optogenetics.">Targeting neurons and photons for optogenetics.</a> Nature Neuroscience. 16 (7), 805-815 (2013).</li><li>Marchetto, M. C. N., 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=A+Model+for+neural+development+and+treatment+of+Rett+Syndrome+using+human+induced+pluripotent+stem+cells.">A Model for neural development and treatment of Rett Syndrome using human induced pluripotent stem cells.</a> Cell. 143 (4), 527-539 (2010).</li><li>Asrican, B., 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=Next-generation+transgenic+mice+for+optogenetic+analysis+of+neural+circuits.">Next-generation transgenic mice for optogenetic analysis of neural circuits.</a> Frontiers in Neural Circuits. 7, 160 (2013).</li></ol>

No conflicts of interest declared.

Optogenética fornece precisão temporal e espacial para a ativação de populações de neurônios 13. Em nossos experimentos, todo o campo da objetiva do microscópio foi iluminado por 30 s para foto-estimular apenas neurônios corticais que expressam ChR2. Isso nos permitiu determinar se conexões sinápticas foram formados entre diferentes populações de neurônios dentro do co-cultura. Nossos resultados revelaram que fotoestimulação aumenta freqüência PSC em neurônios iPSC derivados, o que demonstra que esses neurônios recebem a entrada sináptica dos neurônios corticais pré-sinápticos que expressam ChR2. Este, por sua vez, demonstrado que os neurónios iPSC derivados incorporados com sucesso em circuitos com os neurónios corticais. Existem vários passos críticos para a geração deste sistema de co-cultura. É importante assegurar que as culturas de astrócitos de rato NPC-derivados são saudáveis, com a morte celular mínima, antes de prosseguir com chapeamentode outros tipos de células. Os neurônios corticais e NPCs humanos atribuem bem em astrócitos saudáveis ​​e registros eletrofisiológicos dependem fortemente das condições de cultivo. As outras etapas fundamentais neste protocolo são a eletroporação e chapeamento de neurônios corticais em culturas de astrócitos. Como um grande número de células morrem após a electroporação, é importante para assegurar que, pelo menos, 6 milhões de neurónios corticais são electroporadas para placas com as culturas de astrócitos em placas de 24 poços. Temos observado que, quando menos do que 6 milhões de células foram electroporadas, o número de neurónios que sobrevivem não formar uma rede neuronal densa, que afectou registos electrofisiolicos. O número de neurônios corticais eletroporados também deve ser consistente para cada experimento co-cultura para permitir a comparação entre os diferentes estudos. Para todas as etapas que envolvem a digestão enzimática, é essencial para não deixar as células em solução digestivo durante muito tempo, pois pode levar à morte da célula. Esteabordagem pode ser utilizada para rastrear a capacidade de neurónios derivados de fibroblastos específicos do paciente para formar contactos sinápticos com outros neurónios. Os neurónios derivados de fibroblastos de pacientes que sofrem da perturbação do neurodesenvolvimento síndrome de Rett (RTT) apresentam defeitos de transmissão sináptica: RTT neurónios apresentam uma redução significativa na frequência e amplitude de PSC comparada com os neurónios saudáveis, presumivelmente devido à menor conectividade sináptica 14. Nosso sistema de co-cultura permite o exame dos efeitos da droga sobre os neurônios que apresentam defeitos de desenvolvimento. Isto pode ser realizado por meio de adição de compostos de interesse durante a semeadura de NPCs humanos doentes, bem como durante a exposição contínua a compostos ao longo do tempo de diferenciação neuronal. Embora este sistema de co-cultura pode também ser utilizada como um modelo para o teste da droga, uma limitação desta abordagem é que o processo de investigar os efeitos de cada composto candidato pode ser longo e tedioso quantonão é um método de rastreio de alto rendimento. Após o tratamento com compostos candidatos, os neurónios derivados de iPSC tem que ser corrigido individualmente para determinar os efeitos de cada composto sobre PSCs. Além disso, atualmente há não muitos fibroblastos disponíveis e iPSCs de pacientes. Portanto, será de interesse em um futuro próximo para ter mais células do paciente e também para adaptar este protocolo para triagem de alto rendimento. Por exemplo, por marcação de neurónios iPSC derivados com um indicador de actividade, tais como sensores geneticamente codificados de iões, ou o potencial de membrana, seria possível aumentar a taxa de produção substancialmente pelo procedimento electrofisiológico demorado-pisar lado. Como todo o processo de gerar este sistema de co-cultura, a partir de fibroblastos de reprogramação para executar registos electrofisiolicos, requer mais do que 90 dias, recomenda-se que tanto os iPSCs humanos ou NPCs ser expandida, após a reprogramação e etapas de indução neural respectivamente,e criopreservadas para reduzir o tempo necessário para experiências futuras. Em nossos experimentos, nós expressamos ChR2 em neurônios corticais sob o promotor synapsin1. Porque este promotor é pan-neuronal, que presumivelmente foram photostimulating ambos os neurônios corticais inibitórios e excitatórios. Se o objectivo de experiências futuras é interrogar especificamente circuitos ou inibitórios ou excitatórios, promotores mais específicos podem ser usados. Como alternativa, os neurônios corticais poderia ser obtido a partir de transgênicos (ou injetada-virus ratos) onde a expressão ChR2 é especificamente orientada para as subpopulações geneticamente definidos de neurônios. Por exemplo, os tecidos corticais colheita de um rato que tem Cre recombinase expressa em interneurons contendo parvalbumina (Interneurônios PV) que é cruzada com um mouse com floxed ChR2 renderá uma situação em que os únicos neurônios corticais que expressam ChR2 será interneurons PV 15. O uso de tais ChR2-expressando interneurónios no nosso pedido co-cuLTURE sistema permitirá a determinação de se um neurônio iPSC derivado remendada é capaz de incorporar em um circuito com interneurons PV. Com base em um estudo anterior 9, os neurônios corticais são maduros com sobreposição de dendrites após 14 dias in vitro. Assim, se fotoestimulação não desencadeia uma resposta de um neurónio iPSC derivado de patched, deve indicar que o neurónio não tem a capacidade para fazer ligações sinápticas com neurónios corticais. Um neurônio iPSC derivada é capaz de receber a entrada sináptica ou de outros neurônios iPSC derivados ou neurônios corticais. Se foram utilizadas gravações de sujeição do emplastro tradicional, não seria possível distinguir onde a entrada sináptica é proveniente. A vantagem do nosso sistema é que as respostas pós-sinápticos de entrada presynaptic cortical pode ser evocado seletivamente e identificados. Os procedimentos descritos aqui fornecer uma abordagem simples para avaliar a capacidade dos neurônios iPSC derivados de integrarnuma rede neuronal definido.

Nos últimos anos, a nossa compreensão do neurônio e função do circuito neuronal foram revolucionou por várias ferramentas Optogenetic que controlam a excitabilidade neuronal. Uma dessas ferramentas é o canal de cátions ativado por luz, channelrhodopsin-2 (ChR2): neurônios que expressam ChR2 potenciais de ação fogo em resposta à estimulação de luz 1-3. Porque os neurónios não são normalmente sensíveis à luz, esta capacidade notável abre novos caminhos para o controlo temporal e espacial exacta da actividade de populações geneticamente definidas de neurónios 4 e tem estudos da função cerebral grandemente acelerada. ChR2 foi aplicada a investigação em células estaminais para fins diferentes, de acompanhamento do desenvolvimento neuronal 5 a atividade de redes neurais 6 regulação. Aqui usamos ChR2 para aumentar a utilidade de um sistema de co-cultura neuronal. Sistemas de co-cultura permitir a avaliação das interações entre os diferentes tipos de células.Co-culturas de neurónios e células da glia, os principais tipos de células do sistema nervoso, são comumente usados ​​para investigar a sinalização entre estes tipos de células diferentes, bem como para avaliar a significância do presente sinalização para respostas fisiológicas, a propagação de danos e para examinar o mecanismos moleculares que estão potencialmente envolvidos no presente sinalização 7. Um estudo prévio revelou que iPSCs humanos diferenciar-se em neurónios muito rapidamente na presença de meio de cultura contendo os neurónios corticais de rato primários, astrócitos, oligodendrócitos, microglia e 8. Neste protocolo, demonstramos um método que utiliza ChR2 em um sistema de co-cultura para interrogar conexões sinápticas entre os neurônios e neurônios corticais de rato derivadas de células-tronco pluripotentes induzidas (iPSCs humanos). Nós descrevemos etapas para dissecar e colheita tecidos cerebrais 9, bem como para manter e diferenciar as células progenitoras neurais de rato (NPCs) em astrócitos e NPCs humanos intO neurónios para criar o sistema de co-cultura. Para estabelecer este sistema de co-cultura, foi utilizado o método de reprogramação sem integração descrito por Okita et al. 10 para gerar iPSCs humanos. Estes foram, em seguida, de forma eficiente iPSCs diferenciadas em NPCs em condições quimicamente definidas utilizando inibidores de moléculas pequenas, tal como descrito por Li et al. 11 Na co-culturas, as diferentes populações de neurónios podem ser identificados através de detecção de expressão ChR2 em neurónios corticais e de marcação de neurónios iPSC derivados através da proteína fluorescente, em tandem dímero de tomate (tdTomato). Combinando registos electrofisiolicos de neurónios iPSC derivados com fotoestimulação optogenetic dos neurónios corticais permite avaliar a capacidade destes dois tipos de neurónios para formar circuitos uns com os outros. Especificamente, fotoestimulação de ChR2 expressando neurônios corticais evocar respostas sinápticas em neurônios iPSC derivados que se formaram circuitos sinápticoscom a rede de neurônios corticais photostimulated. Nós demonstramos que o aumento correntes pós-sinápticos são realmente detectado em neurônios iPSC derivados sob tais condições. Este protocolo permite a avaliação de circuitos sináptica sob uma variedade de condições experimentais, incluindo recapitulação dos diferentes modelos de doenças neurológicas usando iPSCs derivadas a partir de fibroblastos de pacientes com a doença.

an optogenetic approach for assessing formation of neuronal connections in a co-culture system

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

Aqui, um protocolo descrevendo os vários passos envolvidos na geração de uma co-cultura consistindo astrócitos, os neurónios corticais e neurónios humanos é ilustrado. IPSCs humano foram obtidas através da reprogramação de fibroblastos utilizando vectores epissomais 10 e especificada na linhagem neural com um cocktail de inibidores de moléculas pequenas, tornando NPCs 11 que foram mantidas a uma densidade elevada (Figura 1A). Vários passos são necessários para gerar a co-cultura: diferenciação dos NPCs do mouse em astrócitos, chapeamento de neurônios corticais de ratos que expressam ChR2, e semeadura de NPCs humanos marcados com tdTomato (Figura 1B). No dia 2 de diferenciação, proteína ácida fibrilar glial (GFAP) + astrócitos podem ser facilmente observadas nas nossas culturas (Figura 1C). Rato NPCs foram deixadas diferenciar durante pelo menos uma semana antes do plaqueamento neurónios corticais que expressam ChR2 sobre a monocamada de astrócitos (Figura 1D). Após 34 semanas de diferenciação NPC humano, tdTomato + neurónios foram detectados nas culturas (Figura 1E). Este sistema de co-cultura permite a detecção de correntes coerentes de neurónios pós-sinápticos iPSC derivados individuais em resposta à estimulação luminosa (Figura 2A). Quando estimulada por luz, potenciais de acção foram gerados em neurónios corticais que expressam ChR2 (Figura 2B). iPSC-neurónios derivados também são excitáveis ​​(Figura 2C), que mostra o aumento da acção de disparo potencial como a amplitude de despolarização impulsos de corrente foi aumentada (Figura 2D). Assim, estas células exibem propriedades neuronais maduros. Na ausência de luz, os neurónios derivados de iPSC recebido entradas sinápticas espontâneas (Figura 2E). Estas correntes dentro pós-sinápticos foram predominantemente mediado por receptores AMPA, porque as correntes através de receptores GABA seria ida e receptores NMDA não carregam uma correnteta segurando potencial de -70 mV. Pelo menos alguns desses insumos eram de neurônios corticais pré-sinápticos que expressam ChR2, porque a estimulação de luz aumentou consideravelmente a frequência das correntes pós-sinápticos (Figura 2E). O tempo de este aumento na entrada sináptica é mostrado na Figura 2F: fotoestimulação dos neurônios corticais foi sustentado ao longo do 30 seg longo luz do flash. Embora a frequência das unidades de atendimento foi elevada quando ChR2 expressando neurônios corticais foram photostimulated (Figura 2G), a sua amplitude não foi afetada (Figura 2H). Em resumo, estes resultados indicam que os neurônios iPSC derivados exibiu propriedades neuronais maduras e poderia formar conexões sinápticas com neurônios corticais pré-sinápticos. <img alt="Figura 1" src="/files/ftp_upload/52408/52408fig1highres.jpg" /> Figura 1: diagramas esquemáticos para a geração de co-cultura(A) Timeline sistema. para a reprogramação fibroblastos humanos em iPSCs e indução neural para NPCs. (B) Timeline para diferenciação terminal de NPCs humanos em neurônios maduros sobre corticais neurônios e astrócitos culturas. (C) rato NPCs diferenciar rapidamente em GFAP + astrócitos após 2 dias in vitro. (D) Após uma semana, os neurónios corticais que expressam ChR2 foram plaqueadas em astrócitos GFAP +. (E) com 4 a 5 semanas após a diferenciação de NPCs humanos, registos electrofisiolicos foram efectuados em tdTomato + neurónios. Barras de escala representam 100 um. <img alt="Figura 2" src="/files/ftp_upload/52408/52408fig2highres.jpg" /> Figura 2: Análise Optogenetic de conectividade sináptica funcional. (A) derivado de células iPSC marcados com tdTomato (vermelho) foram co-cultivadas com os neurónios corticais que expressa o activado pela luzcanal, ChR2 (verde). Gravações de neurônios iPSC derivados revelou atuais (PSC) respostas pós-sinápticos, o que poderia vir de qualquer neurônios corticais ou outros neurônios iPSC derivados. (B) Iluminação (barra azul) provocou uma série de potenciais de ação em neurônios corticais que expressam ChR2. ( células C) iPSC derivados são evocadas por injecção de corrente. (D) Firing vs curva de corrente de células iPSC derivados. (E) fotoestimulação dos neurônios corticais ChR2 expressando (barra azul) aumentou a freqüência de unidades de atendimento em neurônios iPSC derivadas . Claro que (F) Hora do aumento na freqüência PSC produzido por fotoestimulação. Barra azul indica o tempo de fotoestimulação. (G, H) Enquanto frequência CPS foi aumentada por fotoestimulação (G), a amplitude do PSC não foi afectada (H). * Indica p &lt;0,05 comparado com o controlo com o teste t de estudante.

Watch this Scientific Journal Video about An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System at JoVE.com

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Uma Abordagem Optogenetic para Avaliar Formação das conexões neuronais em um sistema de co-cultura

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

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Developmental Biology

13.3K visualizações.  Duke-NUS Graduate Medical School. A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2. 

Vídeo: An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

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

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

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Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Abordagem precisa de ablação celular para modelagem de lesão renal aguda no desenvolvimento de peixe-zebra

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Uma abordagem padronizada para a purificação multiespecífica de células germinais masculinas de mamíferos por dissociação mecânica do tecido e citometria de fluxo

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

O ensaio de co-cultura anel aórtico: Uma ferramenta conveniente para avaliar o potencial angiogênico das células estromais mesenquimais em Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Um modelo In Vitro de um sistema de perfusão paralelo-placa para estudar a aderência bacteriana para enxertar tecidos

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Sistema tridimensional do ensaio da angiogênese usando spheroids da co-cultura dados forma pela colônia endothelial que dá forma a pilhas e às pilhas de haste mesenchymal

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Um método de co-cultura para estudar o crescimento da neurita em resposta aos sinais de paracrina da polpa dentária

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Um sistema otimizado de O9-1/Hidrogel para estudar sinais mecânicos em células de crista neural

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Expressão GFP induzida pela luz em embriões de zebrafish usando o sistema OPtogenético TAEL/C120

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...