Os autores gostariam de reconhecer a Cairn Research Ltd. por sua gentil contribuição financeira que cobria os custos de produção desta publicação. Além disso, agradecemos à Sra. Ines Mueller e à Sra. Stefanie Kestel pelo excelente apoio técnico.
A pesquisa dos autores é apoiada pelo Centro Alemão de Pesquisa Cardiovascular (DZHK), o Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, VO 1568/3-1, IRTG1816 RP12, SFB1002 TPA13 e sob a Estratégia de Excelência da Alemanha - EXC 2067/1- 390729940) e o Else-Kröner-Fresenius Stiftung (EKFS 2016_A20).

1. Preparações celulares
NOTA: Os iPSCs humanos utilizados neste protocolo foram derivados de doadores saudáveis e diferenciados em monocamadas utilizando modulação de moléculas pequenas totalmente definidas de técnicas de sinalização e purificação de lactatos, conforme descrito anteriormente12,13,14. os iPSC-CMs foram mantidos a cada 2-3 dias com um meio de cultura descrito abaixo.
<ol><li>Prepare um meio de cultura de meio basal (RPMI 1640) e 2% de suplemento (B27). Armazenar a 4 °C. Use à temperatura ambiente (RT).</li>
	<li>Prepare um meio de revestimento de meio basal (RPMI 1640), 2% de suplemento (B27) e 1:2000 INibidor ROCK. Armazenar a 4 °C. Use em RT.</li>
	<li>Cubra as tampas de vidro redondo #0 de 10 mm com 150 μL de matriz de membrana de porão livre de fator 1:60 e incubar a 4 °C por 4 h. NOTA: A otimização do volume do deslizamento de tampas é necessária para garantir que todo o vidro esteja coberto, mantendo a tensão adequada da superfície para evitar derramamentos. Recomenda-se 150 μL para tampas redondas de 10 mm. Tamanho do lote, tipo de deslizamento de cobertura, volume de deslizamento e tipo de placa de cultura podem ser adequados às necessidades dos experimentadores.</li>
	<li>Inicie a dissociação iPSC-CM com um reagente de dissociação celular baseado em EDTA. Certifique-se de que a monocamada esteja completamente separada, lavando suavemente com uma pipeta de 1.000 μL.</li>
	<li>Transfira a suspensão celular para um tubo de 15 mL e adicione o meio de revestimento de duplo volume. Centrífuga por 10 min a 100 x g.</li>
	<li>Resuspense a pelota com um volume desejado (volume de ressuspensão) de meio de revestimento. Conte células manualmente ou eletronicamente.</li>
	<li>Selecione a densidade ideal por deslizamento de cobertura (15.000) que permitirá a análise celular isolada mais tarde.</li>
	<li>Calcule o volume de suspensão celular 'ativa' (A) necessária para emplacar todas as tampas nesta densidade desejada. Aplique a seguinte fórmula e retire-se em um tubo separado: <img alt="Equation 1" src="/files/ftp_upload/61890/61890equ01v2.jpg">
</li>
	<li>Calcule o volume de meio de revestimento extra (B) necessário para acomodar cada deslizamento de cobertura em um volume desejado. Aplique a seguinte fórmula e adicione o volume resultante ao tubo de suspensão ativa: <img alt="Equation 2" src="/files/ftp_upload/61890/61890equ02v2.jpg">
</li>
	<li>Remova a matriz das manchas e aplique o 'volume de deslizamento de cobertura' da suspensão celular (A+B) em cada deslizamento de cobertura. Resuspensar regularmente no tubo para garantir até mesmo a distribuição celular.</li>
	<li>Incubar a 37 °C por 1 h. Encha suavemente o poço com meio de revestimento.</li>
	<li>Após 24h, troque mídia com meio de cultura normal e mantenha a cada 2-3 dias.</li>
</ol>2. Configuração experimental
<ol><li>Equipar um microscópio de epifluorescência invertido com uma ampliação de 40x, lente de alta abertura numérica (N.A: > 0,75) para realizar experimentos.</li>
	<li>Junte um LED branco quente de comutação rápida para a porta de iluminação transmitida do microscópio. Insira um simples filtro vermelho de 660 nm no caminho da luz transmitida. NOTA: Esta luz pode ser ativada durante experimentos de fotometria para observar a amostra sem contaminar o sinal fluorescente verde.</li>
	<li>Monte uma cabeça LED de 470 nm de comutação rápida para gravação de fotometria. Insira um filtro de excitação 470/40 na porta epifluorescente do microscópio para limpar a luz gerada pelo LED. NOTA: Para a quantificação ideal de sinal é recomendado um sistema de iluminação com controle de feedback de alta velocidade da saída óptica.</li>
	<li>Insira um cubo de microscópio contendo um divisor de feixe de passagem de 495 nm no carrossel da unidade espelho dentro do microscópio.</li>
	<li>Encaixe um braço de detecção contendo um diafragma de campo ajustável na porta de montagem C do microscópio para permitir a seleção da região de interesse.</li>
	<li>Separadamente, acasalar um detector de fotomultiplier (PMT) e uma câmera USB no microscópio. Isso formará a base do sistema de detecção de emissões.</li>
	<li>Insira um cubo de filtro contendo um divisor de feixe de passagem de 565 nm de comprimento e um filtro de emissão 535/50 na porta PMT. Isso divide a luz de emissão entre os dois detectores. NOTA: Uma câmera conectada à porta transmitida do sistema de detecção de emissões pode detectar luz transmitida sob campo brilhante durante todos os experimentos.</li>
	<li>Acoplam o PMT a uma fonte de alimentação e um amplificador PMT. Conecte a saída do amplificador PMT a um pino de entrada analógico de um sistema de aquisição de dados.</li>
	<li>Filtrar dados analógicos do PMT a 1 kHz ou superior.</li>
	<li>Digitalize os dados em uma frequência que seja pelo menos o dobro da maior frequência presente no sinal analógico (2 kHz ou superior) para cumprir os critérios de Nyquist e evitar o aliasing.</li>
</ol>3. Carregamento celular com VF2.1Cl
NOTA: Todas as etapas que envolvam este corante devem ser realizadas em condições de baixa luz.
<ol><li>Prepare uma solução de banho de Tyrode (em mM): 140 NaCl, 10 HEPES, 10 Glicose, 4 KCl, 1 MgCl2, 2 CaCl2, pH = 7,35 e quente a 37 °C.</li>
	<li>Prepare uma alíquota de solução de carregamento em um tubo de microcentrifuuge misturando 5 μL de 1.000x VF2.1Cl e 50 μL de solução poloxamer solubilizadora de 20%.</li>
	<li>Aplique 5 μL da solução de carregamento a 5 mL de solução de Tyrode aquecida (concentração total de 0,1x de corante) em uma placa de Petri de 20 mm. NOTA: A concentração final de corante é de 0,1x. Este é o 1/10sugerido pelo fabricante. Isso conserva recursos, garante citotoxicidade insignificante e, importante, ainda retém sinais ópticos claros de células carregadas com altas relações de sinal a ruído.</li>
	<li>Adicione um único deslizamento de tampa iPSC-CM ao prato e incubar a 37 °C por 20 min.</li>
	<li>Monte uma câmara de imagem de células vivas aquecidas. Monte no palco do microscópio e preencha com 500 μL de solução fresca de Tyrode.</li>
	<li>Lave o deslizamento com a solução de Tyrode fresco a 37 °C.</li>
	<li>Aplique cuidadosamente a tampa iPSC-CM na câmara de banho pré-aquecida usando fórceps de ponto fino. NOTA: Certifique-se de que a câmara e seu conteúdo estão sempre aquecidos a temperaturas fisiológicas. Se desejar, comece com perfusão contínua da solução aquecida de Tyrode.</li>
</ol>4. Estimulação elétrica do campo
NOTA: O acionamento externo do iPSC-CM é opcional, mas útil para a padronização da dinâmica celular e parâmetros experimentais. Aumenta a facilidade de análise e permite a investigação de efeitos dependentes da frequência.
<ol><li>Conecte uma inserção de estimulação com dois eletrodos de platina espaçados 5 mm de distância na câmara de gravação.</li>
	<li>Conecte um estimulador externo à inserção de estimulação. Definido para 5 ms pulsos de campo bipolar a 0,5 Hz.</li>
	<li>Determine a tensão de estimulação ideal aumentando o estímulo de 1 V para cima. O estímulo limiar é definido como a menor tensão na qual as células começam a se contrair. Aplique tensões cerca de 25% acima deste limite. A faixa normal é entre 1 V e 30 V.</li>
	<li>Corrija a frequência de estimulação com o estimulador externo ou ativá-lo com software de aquisição.</li>
</ol>5. Aquisição potencial de ação óptica
NOTA: Este protocolo utiliza um software comercial para aquisição e análise.
<ol><li>Visualize os miócitos na vista brightfield usando o caminho de luz transmitido e a câmera USB.</li>
	<li>Selecione uma célula isolada e corte firmemente seu caminho óptico com o diafragma de campo garantindo que apenas a luz da célula de interesse seja monitorada.</li>
	<li>Ative o amplificador PMT e defina a fonte PMT para 750 V.</li>
	<li>Execute o protocolo de estimulação (ver passo 4) juntamente com o software de aquisição e ative simultaneamente a luz de excitação de 470 nm. Este último pode ser feito através de um painel remoto ou automatizado a uma intensidade fixa (sinal TTL).</li>
	<li>Ajuste o ganho e o deslocamento do amplificador PMT para garantir que o sinal não satura e seja otimizado para a faixa de detecção do sistema de gravação.</li>
	<li>Recorde de 10 varreduras garantindo que potenciais de ação estáveis sejam detectados.</li>
	<li>Continue gravando e mova imediatamente o estágio do microscópio para adquirir brevemente o sinal de fundo de uma região desprovida de células. Desligue a luz de excitação. NOTA: Este valor de fundo(F offset ) será usado para responder por qualquer fluorescência de fundo.</li>
	<li>Se desejar, perfuse localmente perfuse drogas como 1 μM nifedipine para identificar respostas celulares à manipulação farmacológica.</li>
	<li>De forma sequencial, repita os passos 5.2-5.7, cada vez selecionando uma nova célula. Troque as tampas se desejar para garantir um alto volume de negócios experimental em uma única sessão. NOTA: Os protocolos de carregamento e aquisição de imagens estão descritos na Figura 2.</li>
</ol>6. Análise de dados
<ol><li>Abra uma gravação salva com o software de análise e uma média de 10 varreduras contendo potenciais de ação estimulados de uma única célula.</li>
	<li>Pegue uma média do sinal da linha de base representando Fdeslocamento e subtraia isso a partir do traço médio.</li>
	<li>Calcule ∆F/F0 com a seguinte fórmula (onde F é medido fluorescência e F0 é fluorescência diastólica): <img alt="Equation 3" src="/files/ftp_upload/61890/61890equ03v2.jpg">
</li>
	<li>Identifique a linha de base de rastreamento (diastólica) e a área de interesse (AP) e meça os parâmetros potenciais de ação cardíaca desejados. Isso inclui, mas não se limita ao tempo de decadência para 50% (APD50) e 90% (APD90) de repolarização.</li>
	<li>Exporte os dados desta única célula para um software de planilha.</li>
	<li>Repita as etapas 6.1 – 6.5 para todas as gravações. Avalie os resultados com testes não pagos adequados ou análises de variância.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61890/61890fig2v2.jpg"> Figura 2: Protocolos de carregamento e aquisição de imagens. (A) Gráfico de fluxo do protocolo completo de carregamento VF2.1Cl para iPSC-CMs e cardiomiócitos nativos. (B) Um esquema simplificado de configurações de divisor de feixe (BS) e filtro utilizado neste protocolo para excitação e detecção de emissão de VF2.1Cl em resposta a alterações na tensão transmembrana. <a href="https://www.jove.com/files/ftp_upload/61890/61890fig2v2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

<ol>
	<li>Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+fluorescent+voltage+indicators+for+studying+membrane+potential.">Small molecule fluorescent voltage indicators for studying membrane potential.</a> Current Opinion in Chemical Biology. 33, 74-80 (2016).</li><li>Liang, P., 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=Drug+screening+using+a+library+of+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+reveals+disease-specific+patterns+of+cardiotoxicity.">Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.</a> Circulation. 127 (16), 1677-1691 (2013).</li><li>Horv&#225;th, 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=Low+resting+membrane+potential+and+low+inward+rectifier+potassium+currents+are+not+inherent+features+of+hiPSC-derived+cardiomyocytes.">Low resting membrane potential and low inward rectifier potassium currents are not inherent features of hiPSC-derived cardiomyocytes.</a> Stem Cell Reports. 10 (3), 822-833 (2018).</li><li>Salama, G., Morad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merocyanine+540+as+an+optical+probe+of+transmembrane+electrical+activity+in+the+heart.">Merocyanine 540 as an optical probe of transmembrane electrical activity in the heart.</a> Science. 191 (4226), 485-487 (1976).</li><li>Hortigon-Vinagre, M., 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=The+use+of+ratiometric+fluorescence+measurements+of+the+voltage+sensitive+dye+Di-4-ANEPPS+to+examine+action+potential+characteristics+and+drug+effects+on+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">The use of ratiometric fluorescence measurements of the voltage sensitive dye Di-4-ANEPPS to examine action potential characteristics and drug effects on human induced pluripotent stem cell-derived cardiomyocytes.</a> Toxicological Sciences. 154 (2), 320-331 (2016).</li><li>Blinova, 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=International+multisite+study+of+human-induced+pluripotent+stem+cell-derived+cardiomyocytes+for+drug+proarrhythmic+potential+assessment.">International multisite study of human-induced pluripotent stem cell-derived cardiomyocytes for drug proarrhythmic potential assessment.</a> Cell Reports. 24 (13), 3582-3592 (2018).</li><li>Miller, E. 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=Optically+monitoring+voltage+in+neurons+by+photo-induced+electron+transfer+through+molecular+wires.">Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires.</a> Proceedings of the National Academy of Sciences. 109 (6), 2114-2119 (2012).</li><li>Bedut, S., 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-throughput+drug+profiling+with+voltage-+and+calcium-sensitive+fluorescent+probes+in+human+iPSC-derived+cardiomyocytes.">High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes.</a> American Journal of Physiology-Heart and Circulatory Physiology. 311 (1), 44-53 (2016).</li><li>McKeithan, W. L., 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=An+automated+platform+for+assessment+of+congenital+and+drug-induced+arrhythmia+with+hiPSC-derived+cardiomyocytes.">An automated platform for assessment of congenital and drug-induced arrhythmia with hiPSC-derived cardiomyocytes.</a> Frontiers in Physiology. 8, 766 (2017).</li><li>Duncan, 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=Drug-mediated+shortening+of+action+potentials+in+LQTS2+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Drug-mediated shortening of action potentials in LQTS2 human induced pluripotent stem cell-derived cardiomyocytes.</a> Stem Cells and Development. 26 (23), 1695-1705 (2017).</li><li>Asakura, K., Hayashi, S., Ojima, A., Taniguchi, T., Miyamoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+acquisition+and+analysis+methods+in+multi-electrode+array+experiments+with+iPS+cell-derived+cardiomyocytes.">Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes.</a> Journal of Pharmacological and Toxicological Methods. 75, 17-26 (2015).</li><li>Lian, X., 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=Directed+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+by+modulating+Wnt/&#946;-catenin+signaling+under+fully+defined+conditions.">Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/&#946;-catenin signaling under fully defined conditions.</a> Nature Protocols. 8 (1), 162-175 (2013).</li><li>Burridge, P. 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=Chemically+defined+generation+of+human+cardiomyocytes.">Chemically defined generation of human cardiomyocytes.</a> Nature methods. 11 (8), 855-860 (2014).</li><li>Kleinsorge, M., Cyganek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-directed+differentiation+of+human+iPSCs+into+atrial+and+ventricular+cardiomyocytes.">Subtype-directed differentiation of human iPSCs into atrial and ventricular cardiomyocytes.</a> STAR Protocols. , 100026 (2020).</li><li>Knollmann, B. C., Katchman, A. N., Franz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monophasic+action+potential+recordings+from+intact+mouse+heart:+validation,+regional+heterogeneity,+and+relation+to+refractoriness.">Monophasic action potential recordings from intact mouse heart: validation, regional heterogeneity, and relation to refractoriness.</a> Journal of Cardiovascular Electrophysiology. 12 (11), 1286-1294 (2001).</li><li>Leopold, J. A., Loscalzo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+precision+medicine+in+cardiovascular+disease.">Emerging role of precision medicine in cardiovascular disease.</a> Circulation Research. 122 (9), 1302-1315 (2018).</li><li>Voigt, N., Zhou, X. B., Dobrev, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+atrial+myocytes+for+simultaneous+measurements+of+Ca2++transients+and+membrane+currents.">Isolation of human atrial myocytes for simultaneous measurements of Ca2+ transients and membrane currents.</a> Journal of Visualized Experiments. (77), e50235 (2013).</li><li>Voigt, 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=Enhanced+sarcoplasmic+reticulum+Ca2++Leak+and+increased+Na+-Ca2++exchanger+function+underlie+delayed+afterdepolarizations+in+patients+with+chronic+atrial+fibrillation.">Enhanced sarcoplasmic reticulum Ca2+ Leak and increased Na+-Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation.</a> Circulation. 125 (17), 2059-2070 (2012).</li><li>Voigt, 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=Cellular+and+molecular+mechanisms+of+atrial+arrhythmogenesis+in+patients+with+paroxysmal+atrial+fibrillation.">Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation.</a> Circulation. 129 (2), 145-156 (2014).</li><li>Fakuade, F. E., 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=Altered+atrial+cytosolic+calcium+handling+contributes+to+the+development+of+postoperative+atrial+fibrillation.">Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation.</a> Cardiovascular Research. , 162 (2020).</li><li>Gross, E., Bedlack, R. S., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-wavelength+ratiometric+fluorescence+measurement+of+the+membrane+dipole+potential.">Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.</a> Biophysical Journal. 67 (1), 208-216 (1994).</li><li>Matiukas, 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=Near-infrared+voltage-sensitive+fluorescent+dyes+optimized+for+optical+mapping+in+blood-perfused+myocardium.">Near-infrared voltage-sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium.</a> Heart Rhythm. 4 (11), 1441-1451 (2007).</li><li>Mutoh, 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=Spectrally-resolved+response+properties+of+the+three+most+advanced+fret+based+fluorescent+protein+voltage+probes.">Spectrally-resolved response properties of the three most advanced fret based fluorescent protein voltage probes.</a> PLoS One. 4 (2), 4555 (2009).</li><li>Hochbaum, D. R., 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=All-optical+electrophysiology+in+mammalian+neurons+using+engineered+microbial+rhodopsins.">All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.</a> Nature Methods. 11 (8), 825-833 (2014).</li><li>Huang, Y. L., Walker, A. S., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photostable+silicon+rhodamine+platform+for+optical+voltage+sensing.">A photostable silicon rhodamine platform for optical voltage sensing.</a> Journal of the American Chemical Society. 137 (33), 10767-10776 (2015).</li><li>Deal, P. E., Kulkarni, R. U., Al-Abdullatif, S. H., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isomerically+pure+tetramethylrhodamine+voltage+reporters.">Isomerically pure tetramethylrhodamine voltage reporters.</a> Journal of the American Chemical Society. 138 (29), 9085-9088 (2016).</li><li>Fluhler, E., Burnham, V. G., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectra,+membrane+binding,+and+potentiometric+responses+of+new+charge+shift+probes.">Spectra, membrane binding, and potentiometric responses of new charge shift probes.</a> Biochemistry. 24 (21), 5749-5755 (1985).</li><li>Fromherz, P., Muller, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+fluorescence+of+amphiphilic+hemicyanine+dyes+in+neuron+membrane.">Voltage-sensitive fluorescence of amphiphilic hemicyanine dyes in neuron membrane.</a> Biochimica et Biophysica Acta. 1150 (2), 111-122 (1993).</li><li>Salama, 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=Properties+of+new,+long-wavelength,+voltage-sensitive+dyes+in+the+heart.">Properties of new, long-wavelength, voltage-sensitive dyes in the heart.</a> Journal of Membrane Biology. 208 (2), 125-140 (2005).</li><li>Jin, L., 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=Single+action+potentials+and+subthreshold+electrical+events+imaged+in+neurons+with+a+fluorescent+protein+voltage+probe.">Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.</a> Neuron. 75 (5), 779-785 (2012).</li><li>Kralj, J. M., Douglass, A. D., Hochbaum, D. R., MacLaurin, D., Cohen, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+action+potentials+in+mammalian+neurons+using+a+microbial+rhodopsin.">Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.</a> Nature Methods. 9 (1), 90-95 (2012).</li><li>Tsutsui, H., Karasawa, S., Okamura, Y., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+membrane+voltage+measurements+using+FRET+with+new+fluorescent+proteins.">Improving membrane voltage measurements using FRET with new fluorescent proteins.</a> Nature Methods. 5 (8), 683-685 (2008).</li><li>Lundby, A., Mutoh, H., Dimitrov, D., Akemann, W., Kn&#246;pfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+genetically+encodable+fluorescent+voltage+sensor+exploiting+fast+Ci-VSP+voltage-sensing+movements.">Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements.</a> PLoS One. 3 (6), 2514 (2008).</li><li>Bradley, J., Luo, R., Otis, T. S., DiGregorio, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submillisecond+optical+reporting+of+membrane+potential+in+situ+using+a+neuronal+tracer+dye.">Submillisecond optical reporting of membrane potential in situ using a neuronal tracer dye.</a> The Journal of neuroscience. 29 (29), 9197-9209 (2009).</li><li>Herron, T. J., Lee, P., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+voltage+and+calcium+in+cardiac+cells+&amp;+tissues.">Optical imaging of voltage and calcium in cardiac cells &amp; tissues.</a> Circulation Research. 110 (4), 609-623 (2012).</li><li>Kappadan, V., 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-resolution+optical+measurement+of+cardiac+restitution,+contraction,+and+fibrillation+dynamics+in+beating+vs.+blebbistatin-uncoupled+isolated+rabbit+hearts.">High-resolution optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts.</a> Frontiers in Physiology. 11, 464 (2020).</li><li>Kettlewell, S., Walker, N. L., Cobbe, S. M., Burton, F. L., Smith, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrophysiological+and+mechanical+effects+of+2,3-butane-dione+monoxime+and+cytochalasin-D+in+the+Langendorff+perfused+rabbit+heart.">The electrophysiological and mechanical effects of 2,3-butane-dione monoxime and cytochalasin-D in the Langendorff perfused rabbit heart.</a> Experimental Physiology. 89 (2), 163-172 (2004).</li><li>K&#233;pir&#243;, M., 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=para-Nitroblebbistatin,+the+non-cytotoxic+and+photostable+Myosin+inhibitor.">para-Nitroblebbistatin, the non-cytotoxic and photostable Myosin inhibitor.</a> Angewandte Chemie International Edition. 53 (31), 8211-8215 (2014).</li></ol>

Cairn Research Ltd apoiou esta publicação cobrindo os custos de produção do arquivo de vídeo.

Aqui descrevemos um protocolo básico para adquirir facilmente perfis de AP detalhados de iPSC-CMs isolados adequados para modelagem eletrofisiológica e rastreamento de medicamentos cardíacos. Detectamos APs regulares e robustos de nossos iPSC-CMs pouco semeados, o que sugere a funcionalidade do indicador e a fidelidade metodológica.
Devido ao amplo espectro de metodologias comerciais para reprogramação do iPSC e falta de padronização para protocolos de diferenciação cardíaca, os mod...

A modelagem eletrofisiológica de cardiomiócitos e a construção de plataformas eficientes para rastreamento de medicamentos cardíacos é essencial para o desenvolvimento de estratégias terapêuticas para uma variedade de distúrbios arrítmicos. A rápida expansão da tecnologia de células-tronco pluripotentes induzidas (iPSC) produziu incursões promissoras na modelagem de doenças humanas e na investigação farmacológica usando cardiomiócitos isolados derivados do paciente (iPSC-CM). Técnicas "padrão-ouro" para caracterização eletrofisiológica dessas células através de patch-clamp (grampo atual) podem quantificar o potencial de ação (AP) morfologia e duração, no entanto, este método é incrivelmente complexo e lento, e não é adequado para aquisição de dados de alto rendimento1. IPSC-CMs são regularmente relatados como com um potencial de membrana diastólica aumentada e aumento da corrente de vazamento quando comparados aos cardiomiócitos nativos adultos2. Sugere-se que o tamanho celular menor e a redução da capacitância da membrana observada nos iPSC-CMs podem produzir algum erro sistemático ao usar a técnica de grampo atual, talvez explicando esses desvios3. A fim de maximizar a utilidade de uma plataforma iPSC-CM, um método adicional é valioso para aumentar o throughput e garantir a precisão dos dados ao caracterizar alterações de tensão transmembrana em um único nível celular em iPSC-CMs.
Os corantes sensíveis à tensão (VSD) têm sido há muito tempo um método proposto para fornecer análises mais rápidas, não invasivas e equivalentes da cinética AP cardíaca comparativa com as das técnicas tradicionais4. Um estudo recente demonstrou a adequação da fotometria da sonda sensível à tensão racionmétrica para quantificar com precisão o AP5cardíaco . Além disso, a capacidade de escalar prontamente abordagens de fotometria óptica empresta essa técnica a telas de cardiotoxicidade em larga escala críticas no desenvolvimento de medicamentos terapêuticos (por exemplo, CiPA). O desenvolvimento de protocolos padronizados de cardiotoxicidade em um estudo cego de vários sites usando matriz de microeletrodos e técnicas ópticas de sensoriamento de tensão demonstrou o valor-chave desta abordagem6.
Muitos corantes potencialiométricos estão comercialmente disponíveis, e o desenvolvimento sintético contínuo de novas sondas mostra um potencial empolgante para simplificar sua eficácia em uma variedade de construções cardíacas e neurais. O VSD ideal terá cinética aumentada e sensibilidade, enquanto exibe menor carga capacitiva, fotobleaching e citotoxicidade. O recém-sintetizado VF2.1Cl (FluoVolt) expressa muitas dessas propriedades benéficas em grande parte devido à sua nova estrutura molecular baseada em fios, compartilhada por outros membros da nova família VoltageFluor (VF)7. Em contraste com vsds eletrocrômicos comuns em que sondas simples molecular e eletricamente conjugam à membrana plasmática, este corante consiste em um fio sintético passivamente inserido, de abrangência de membrana, que emparelha um doador rico em elétrons com um fluoróforo de fluoresceína modificada (FITC). Detalhes mecanicistas são fornecidos na Figura 1. Este corante demonstra excelente sensibilidade às flutuações de tensão da membrana, exibindo uma mudança de 27% na intensidade de emissão por 100 mV em oposição a ~10% visto em outras sondas comuns a velocidadescomparáveis 7. Além disso, os sistemas PeT baseados em fios não interagem diretamente com o campo elétrico celular que produz interferência elétrica mínima e mudanças insignificantes na carga capacitiva celular.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61890/61890fig01.jpg"> Figura 1: Parâmetros químicos, espectrais e mecanicistas de corante VF2.1Cl. (A) Estrutura química de VF2.1Cl. Características moleculares a notar incluem múltiplos grupos de alquila dentro do fio molecular de fenileno vinylene que facilitam a inserção na membrana plasmática. Um grupo de ácido sulfônico carregado negativamente conjugado à sonda FITC garante estabilização de fluoróforo na superfície extracelular e auxilia perto da inserção perpendicular em relação ao campo elétrico da bicamada lipídica. (B) Um esquema simplificado de VF2.1Cl perpendicular incorporando na membrana plasmática de uma célula alvo. (C) Espectro de absorção e emissão de corante VF2.1Cl. O espectro é idêntico ao dos testes FITC e GFP padrão. (D) Representação do modo mecanicista de ação de VF2.1Cl. Em condições de repouso (hiperpolarizadas), tensões intracelulares negativas conduzem elétrons livres em direção ao fluoróforo rostral. A abundância de elétrons garante que a transferência de elétrons induzidos por foto (PeT) seja favorecida como um caminho para fora do estado animado após a excitação óptica, efetivamente saciando a fluorescência. Em contraste, um potencial de membrana despolarizada influencia o movimento de elétrons descendentes favorecendo a fluorescência após a excitação óptica. A resposta fluorescente resultante está linearmente relacionada à tensão da membrana e pode ser precisamente utilizada para coletar informações temporais detalhadas sobre cinética eletrofisiológica celular. (E) Representativo brightfield (superior) e fluorescência a 470 nm (inferior) imagens de cardiomiócitos leporina carregados com VF2.1Cl. (F) Z pilha de um único cardiomiócito carregado. As setas indicam áreas de localização clara de VF2.1Cl para a membrana celular. As imagens foram adquiridas com um sistema confocal de disco giratório consistindo de uma cabeça confocal de disco giratório X-lightv3 com um padrão de pinhole de 50 μm; Iluminador LDI-7; Câmera Prime95B e um objetivo PlanApo Lambda 100x. Barra de escala: 20 μm. <a href="https://www.jove.com/files/ftp_upload/61890/61890fig01large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
A sonda FITC conjugada ao VF2.1Cl garante que ele pode ser usado efetivamente sob configurações padrão e filtro GFP, e requer apenas um único sistema de aquisição de canais, ambos características comuns de plataformas de imagem fluorescentes. A análise de monocamadas humanas densas com este corante foi recentemente relatada8,9,10,11. Nosso protocolo difere desses estudos devido à nossa investigação de iPSC-CMs únicos e isolados, imperturbáveis pelas influências elétricas e paracrinas de monocamadas sincicial densas, e pelo uso de um sistema de fotometria acessível e personalizável em oposição a complexos arranjos de imagem confocal ou de campo amplo.
Aqui, descrevemos nosso protocolo para a rápida aquisição e análise de APs ópticos robustos de cardiomiócitos isolados derivados do iPSC e cardiomiócitos nativos (ver Arquivo Suplementar). Usamos VF2.1Cl juntamente com uma plataforma de última geração personalizável para medições de fotometria de célula única. Esses protocolos experimentais foram aprovados pelo comitê de ética do Centro Médico Universitário Göttingen (nº 10/9/15).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Carl Roth&#160;</td>
			<td>HN04.2</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous&#160;BioChemica</td>
			<td>ITW Reagents</td>
			<td>A1422</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt Membrane Potential Kit&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>HN77.4</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamanin</td>
			<td>Sigma-Aldrich</td>
			<td>114956-81-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>21829-25-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen&#160;</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y27632</td>
			<td>Stemolecule</td>
			<td>04-0012-10</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>15040033</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>495LP Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circle Coverslips, Thickness 0</td>
			<td>Thermo Scientific</td>
			<td>CB00100RA020MNT0</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1550B</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual OptoLED Power Supply&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET470/40x Excitation Filter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET535/50m</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etched Neubauer Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Cubes&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IX73 Inverted Microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MonoLED</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiport Adaptors&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myopacer Cell Stimulator&#160;</td>
			<td>IonOptix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optomask Shutter&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optoscan System Controller</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PH-1 Temperature Controlled Platform&#160;&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier Detector&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Amplifier Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Supply Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RC-26G Open Bath Chamber&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SA-OLY/2AL Stage Adaptor&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T565lpxr Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T660lpxr Dichroic Beamsplitter</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC-20 Dual Channel Temperature Controller&#160;</td>
			<td>npi Electronic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UPLFLN 40X Objective</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>USB 3.0 Colour Camera&#160;</td>
			<td>Imaging Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IC Capture 2.4&#160;</td>
			<td>Imaging Source&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes

As técnicas convencionais de microeletrodos intraecelulares para quantificar a eletrofisiologia cardiomiocócica são extremamente complexas, intensivas em mão-de-obra e tipicamente realizadas em baixo rendimento. A rápida e contínua expansão da tecnologia de células-tronco pluripotentes induzidas (iPSC) apresenta um novo padrão em pesquisa cardiovascular e métodos alternativos são agora necessários para aumentar o throughput de dados eletrofisiológicos em um único nível celular. VF2.1Cl é um corante sensível à tensão recentemente derivado que fornece uma resposta rápida de canal único e de alta magnitude às flutuações no potencial da membrana. Possui cinética superior às de outros indicadores de tensão existentes e disponibiliza dados funcionais equivalentes aos das técnicas tradicionais de microeletrodo. Aqui, demonstramos uma caracterização potencial de ação simplificada e não invasiva em cardiomiócitos derivados do iPSC humanos com ritmo externo usando um sistema de fotometria modular e altamente acessível.

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61890/61890fig03.jpg"> Figura 3: Perfis de potencial de ação óptica (AP) de cardiomiócitos nativos isolados e cardiomiócitos pluripotentes induzidos por humanos derivados de células-tronco (iPSC-CM). (A) Ap óptico representativo de um único cardiomiócito murino (centro) com média ± SEM de APD50 e APD90 (n = 7, à direita). (B) Ap óptico represe...

Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Aqui descrevemos a aquisição óptica e a caracterização de potenciais de ação a partir de cardiomiócitos derivados de células-tronco pluripotentes induzidos usando um sistema de fotometria modular de alta velocidade.

Medição potencial de ação óptica unicelular em cardiomiócitos derivados de células-tronco pluripotentes induzidos por humanos

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

single-cell-optical-action-potential-measurement-human-induced

Research

JoVE Journal

Bioengineering

4.0K visualizações.  University Medical Center Goettingen, Goettingen, Germany. Aqui descrevemos a aquisição óptica e a caracterização de potenciais de ação a partir de cardiomiócitos derivados de células-tronco pluripotentes induzidos usando um sistema de fotometria modular de alta velocidade.

Vídeo: Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

Mapeamento de potenciais de ação óptica e transientes de cálcio no coração de rato

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Bioengenharia

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Desenvolvimento, expansão e<em&gt; In vivo</em&gt; Monitorização das células NK humanas de células estaminais embrionárias humanas (hESCs) e e induziu células-tronco pluripotentes (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplante de Progenitores Miogênicos derivados de células-tronco pluripotentes induzidos em modelos de regeneração muscular

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Redes humanas de cardiomiócitos derivadas de iPSC em matrizes de microeletrodos Multipoços para gravações em potencial de ação recorrente

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Diferenciação guiada de podocitos renais maduros de células-tronco pluripotentes induzidas humanas sob condições quimicamente definidas

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chip de glomérulo renal isogênico projetado a partir de células-tronco pluripotentes induzidas por humanos

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Avaliação da Terapia de Modulação da Contratilidade Cardíaca em Cardiomiócitos Derivados de Células-Tronco Humanas 2D

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

Geração de Organoides de Vasos Sanguíneos Humanos a partir de Células-Tronco Pluripotentes

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Geração, Triagem de Alto Rendimento e Biobanco de Esferoides Cardíacos Derivados de Células-Tronco Pluripotentes Induzidos por Humanos.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Triagem de cardiotoxicidade de alto rendimento usando monocamadas de cardiomiócitos derivadas de células-tronco pluripotentes induzidas por humanos maduros

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...