Agradecemos Andrea Parmeggiani (L2C e DIMNP, Montpellier), Benoit Charlot (IES, Montpellier), bem como os membros do laboratório para discussões úteis, Christophe Duperray para obter ajuda com a análise FACS, a instalação de imagem RIO Montpellier (MRI) para fornecer o ambiente adequado para FACS e microscopia confocal. BNM foi apoiado por uma bolsa de pós-graduação do Labex Numev (convenção ANR-10-LabX-20). AB foi apoiado por uma bolsa de estudo de graduação da Universidade de Varsóvia e da União Europeia (n ° POKL.04.01.02-00-221 / 12). MLV é um cientista da equipe do Centro Nacional de Pesquisa Científica (CNRS).

Dia 1 1. Rotulagem da Célula-tronco mesenquimais (MSC) As mitocôndrias (Opcional) <ol><li> Dois dias antes da preparação mitocôndrias, sementes MSCs humanas numa placa de cultura a 100 mm, em 10 ml de aMEM / FBS a 10%, de modo a ter 4 x 10 5 MSCs em cultura no Dia 1. </li><li> Lavar as MSCs com PBS (4 mL) e adicionar 4 ml de aMEM / FBS a 1% (pré-aquecido a 37 ° C). </li><li> Adicionar a quantidade necessária de mitocôndrias corante vital e incubar as células durante 30 minutos na temperatura de 37 ° C incubadora. </li><li> Remover a solução de corante de mitocôndrias, lavar as células duas vezes com 4 ml pré-aquecido (37 ° C) aMEM / FBS a 1% e adicionar 4 ml de volta aMEM / FBS a 10%. Incubar as células a 37 ° C. </li><li> Mudar o meio de cultura (10 ml aMEM / FBS a 10%) após 30 min e, outra vez, duas horas mais tarde. </li></ol> Dia 1 2. Rotulagem dos células de glioblastoma-tronco (SGC) (opcional, consulte seção de discussão) <table border="1" fo:keep-together.within-page = &quot;1&quot; fo: manter-com-next.within-page = &quot;always&quot;&gt; <tbody><tr><td> Meio de cultura celular </td><td> Composição </td></tr><tr><td rowspan="5"> meio basal GSC </td><td> DMEM / F-12 suplementado com </td></tr><tr><td> A insulina 20 mg / ml </td></tr><tr><td> 1x suplemento N2 </td></tr><tr><td> Glicose a 3 g / L </td></tr><tr><td> L-glutamina 2 mM </td></tr><tr><td rowspan="9"> meio de proliferação GSC </td><td> Adicionar ao meio basal: </td></tr><tr><td> 1x suplemento B27 </td></tr><tr><td> EGF 10 ng / mL </td></tr><tr><td> bFGF 10 ng / mL </td></tr><tr><td> Fungine 10 mg / ml </td></tr><tr><td> Fungizona 0,25 mg / ml </td></tr><tr><td> Heparina 2 mg / ml </td></tr><tr><td> Ciprofloxacina 2 ug / ml </td></tr><tr><td> Gentamicina 2 ug / ml </td></tr><tr><td rowspan="4"> meio de proliferação MSC </td><td> &amp; #945; MEM suplementado com </td></tr><tr><td> L-glutamina 2 mM </td></tr><tr><td> 10% de FBS </td></tr><tr><td> bFGF a 2 ng / mL </td></tr></tbody></table> Tabela 1: Meios de Cultura. <ol><li> Dissociar GSCS (linha GB4 célula 32 (10 x 10 6 células) cultivadas como neurospheres no poli-HEMA frascos de cultura de células revestidas (veja as etapas 3.1 a 3.12). </li><li> GSCS semente em uma placa de 48 poços em 10 5 células / poço em meio de proliferação SGC (500 ul) (ver Tabela 1). </li><li> Centrifugar a placa a 270 xg durante 5 min a 20 ° C. </li><li> Adicionar a quantidade necessária de corante vital de células e incubar durante 30 min a 37 ° C. </li><li> Adicionar 500 ul de SGC meio basal (Tabela 1) por poço da placa de 48 poços. </li><li> Centrifugar a placa a 270 xg a 20 ° C durante 5 min. Aspirar o sobrenadante. </li><li> Repita os passos 2,5-2,6. </li><li> Adicionar 500 ul de meio basal SGC e incubar a 37 ° C durante 30 min. </li><li> Centrifugar a placa a 270 xg a 20 ° C durante 5 min. Aspirar o sobrenadante. </li><li> Adicionar 500 ul de meio de proliferação SGC por poço da placa de 48 poços e incuba-se no 37 ° C incubadora. Nota: A quantidade de GSCS indicado (10 X 10 6 células) permite realizar as diferentes experiências de dose-resposta e os controlos de FACS. Uma vez que as condições experimentais são mais precisamente definido esse valor pode ser reduzido. </li></ol> Dia 2 3. Sementeira de Células Estaminais Glioblastoma <ol><li> Recolher as neuroesferas SGC (10 X 10 6 células) por centrifugação a 270 xg durante 5 min, a 20 ° C em um tubo de 50 ml. </li><li> Lavar as células com 5 ml de HBSS, e centrifugar a 270 xg durante 5 min a 20 ° C. </li><li> Aspirar o sobrenadante. </li><li> Suavemente ressuspender o sedimento em 100 ul SGC tripsina-EDTA (0,25%) (por 10 x 10 <s-se&gt; 6 células) </li><li> Incubar a 37 ° C durante 3 min. </li><li> Adiciona-se 10 uL de CaCl2 (20 mM) e 2 ul de ADNase I (10 mg / ml). </li><li> Dissociar as neurospheres cuidado pipetando para cima e para baixo (30-50x) com uma pipeta P200. Evitar bolhas. Verifique sob o microscópio que todos os GSCS são dissociados. </li><li> Adicionar 10 ul de inibidor de tripsina (5%) e 10 ml de HBSS. </li><li> Centrífuga GSCS a 270 xg durante 7 minutos a 20 ° C. </li><li> Descartar o sobrenadante e adicionar 10 ml de SGC meio basal (Tabela 1). </li><li> Contagem GSCS com uma câmara de contagem Thoma e depois centrifugar as células a 270 xg durante 7 minutos a 20 ° C. </li><li> Adicionar o volume apropriado de meio de proliferação SGC (Tabela 1) para alcançar a concentração celular de 6 a 10 GSCS / ml. </li><li> De sementes 10 5 GSCS (100 uL da suspensão de células) por cavidade de uma placa de 96 poços. </li><li> Centrifugar as placas a 270 xg durante 7 minutos a 20 ° C para obter GSCS no botTom de poços. </li><li> Incubar a placa de 96 poços a 37 ° C até que a secção 5. </li></ol> Dia 2 4. MSC mitocôndrias Isolamento <ol><li> Ajustar a temperatura microtubo de centrifugação a 4 ° C. </li><li> Preparar dois tubos de 1,5 ml &quot;A&quot; e &quot;C&quot; que contém os reagentes para a extracção de mitocôndrias (200 ul de reagente A e 400 ul de reagente C, contendo ambos os inibidores da protease isento de EDTA). Prepare 2 outros tubos, rotulados como &quot;MSC&quot; e &quot;Mito&quot;. Mantenha todos os tubos em gelo. Também preparar tubos para as diluições em série mitocôndrias. </li><li> Lavar as MSCs com 10 ml pré-aquecido (37 ° C) PBS. </li><li> Lavar as MSCs com 2 ml de tripsina (sem EDTA) durante 10 segundos e adicionar 1 ml de tripsina (sem EDTA). Incubar as células durante 5-10 min a 37 ° C. </li><li> Recuperar as MSCs pela adição de 10 ml aMEM / FBS 10%, transferência para um tubo de 50 ml. </li><li> células centrifugar a 270 xg durante 5 min a 20 ° C. </li><li> Descartar o sobrenadante e adicionar10 ml de aMEM / FBS a 10% ao sedimento celular. </li><li> Contar as MSCs com uma câmara de contagem Malassez. </li><li> Centrifugar as MSCs (4-5 x 10 5) a 270 xg durante 5 min a 20 ° C. </li><li> Rejeitar o sobrenadante, adicionar 1 M arrefecido com gelo aMEM / FBS a 10% ao sedimento celular e transferência das células para o &quot;MSC&quot; tubo marcado com (preparado no passo 4.2). Mantenha o tubo em gelo. </li><li> Centrifugar o tubo contendo as MSCs em 900 xg durante 5 min, a 4 ° C. </li><li> Remova todas as médias residual do tubo. </li><li> Adicionar 200 ul de mitocôndrias Isolamento Reagente A (contendo os inibidores de protease isento de EDTA). Vortex a velocidade média durante 5 segundos e deixar os tubos em gelo durante exactamente 2 min. </li><li> Adicionar 2,5 mL de mitocôndrias Isolamento Reagente B. Vortex à velocidade máxima por 10 segundos, e depois sair tubos em gelo. Repetir a cada 30 seg durante 5 min. </li><li> Adicionar 200 ul de Reagente de Isolamento As mitocôndrias C (contendo os inibidores de protease isento de EDTA). Misturar por inclinação do tubo (ROughly 30 vezes, não vortex). Centrifuga-se o tubo a 700 xg durante 10 min a 4 ° C. </li><li> Transferir o sobrenadante (contendo o mitocôndrias MSC) para o tubo &quot;Mito&quot; (preparado no passo 4.2). </li><li> Centrifugar a 3000 x g, 15 min a 4 ° C, para obter o sedimento mitocôndrias. Descartar o sobrenadante. O sedimento contem os mitocôndrias isoladas. </li><li> Lavar o sedimento com 200 ul de mitocôndrias do Reagente C. Em seguida, centrifugar a 12000 xg, 5 min a 4 ° C, para obter o sedimento mitocôndrias. </li></ol> 5. Transferência de Isolated MSC mitocôndria para GSCS (MitoCeption) <ol><li> Adicionar 200 ul da (0 ° C) meio de pré-arrefecida SGC proliferação ao sedimento de mitocôndrias isoladas de MSC (4 x 10 5). </li><li> Dilui-se a preparação de mitocôndria (no meio de proliferação SGC) para adicionar consistentemente 20 ul de suspensão mitocondrial para o GSCS. </li><li> Adicionar o volume de mitocôndrias isoladas nas cavidades da 96-WEll placa contendo os GSCS (do passo 3.15), na concentração desejada (0,1 a 10 ug). Adicionar lentamente a mitocôndria, perto do fundo do poço, cobrindo toda a superfície, pelo menos uma vez. </li><li> Para controlar MSC mitocôndrias corante vital vazamento, adicionar as mesmas quantidades de preparação mitocôndrias (0,1 a 10 ug) a poços de uma placa de 96 poços contendo SGC meio de proliferação apenas (sem GSCS) (ver parte 6.2 para a detecção de fugas de corante vital). </li><li> Centrifugar a placa de 96 poços contendo as mitocôndrias GSCS destinatário com a mitocôndria MSC (passo 5.3) e a placa de controlo (passo 5.4) a 1500 xg durante 15 min a 4 ° C. </li><li> Colocar as placas de cultura celular na incubadora a 37 ° C imediatamente após a centrifugação. Nota: O protocolo de transferência de mitocôndrias baseia-se na centrifugação da suspensão de mitocôndrias em células cultivadas na força de centrifugação adequado, com um número de processos de centrifugação que pode ser ajustada dependendo do System células de dador / receptor mitocôndrias. </li></ol> Dia 3 6. Análise da transferência de mitocôndrias por FACS e Imagem Confocal <ol><li> Preparação de amostras para a análise por FACS SGC Nota: Se MSC mitocôndrias foram marcadas com um corante vital de antemão (ponto 1), a eficiência pode ser monitorizada por FACS, 24 horas após a transferência de mitocôndrias. <ol><li> Centrifugar a placa de 96 poços com os GSCS MitoCepted a 270 xg durante 5 min a 20 ° C. </li><li> Descartar o sobrenadante. </li><li> Adicionar 100 ul de tripsina (sem EDTA) em cada poço. Incubar a 37 ° C durante 3 min. Pipeta-se para baixo e para dissociar as neuroesferas formados durante o período de tempo de 24 h. </li><li> Adicionar 100 ul de SGC meio basal. </li><li> Centrifugar a placa a 270 xg durante 5 min a 20 ° C e desprezar o sobrenadante. </li><li> Ressuspender GSCS em 300 ul GSC basal médio e transferência para FACS adaptada tubos. </li><li> Realizar a um FACSANÁLISE. </li></ol></li><li> Controlo para detecção de fugas mitocôndrias corante vital do mitocôndrias isoladas (FACS) Nota: O objetivo deste passo é determinar o sinal de FACS de fundo que não refletem uma real transferência MSC mitocôndrias, mas sim a mera fuga de corante vital da mitocôndria MSC rotulados usado para MitoCeption (passo 5.3). <ol><li> Semente células SGC, tal como descrito nas etapas 3.1 a 3.15. </li><li> Incubar as células a 37 ° C durante 1 h. </li><li> Centrifugar a placa de 96 poços contendo mitocôndrias única (do passo entre 5,4 e 5,6) a 1500 xg durante 5 min a 20 ° C. </li><li> Aspirar o meio de cultura a partir do SGC placa de 96 poços (passo 6.2.2) e substituí-lo por meio incubados com mitocôndrias única (sobrenadante a partir do passo 6.2.3). </li><li> Incubar a placa de 96 poços contendo os GSCS a 37 ° C durante 2 h. </li><li> Proceder como nas etapas 6.1.1 a 6.1.7 para análise FACS. </li></ol></li><li> Preparação do GSC samples de imagem confocal Nota: Se a transferência de MSC mitocôndria para GSCS é para ser analisada por imagiologia de fluorescência, ambas as MSCs e GSCS necessita ser etiquetado de antemão, respectivamente com mitocôndrias (passo 1) e de células (passo 2) corantes vitais. <ol><li> Prepare GSCS como nos passos 6.1.1 a 6.1.5. </li><li> Ressuspender GSCS em 2 ml de meio de proliferação SGC contendo 2% de FBS e as sementes em placas de cultura de 35 mm (com fundo de vidro). </li><li> Executar imagem confocal sobre os GSCS com as mitocôndrias MSC fluorescentes transferidos 24 horas mais tarde. </li></ol></li></ol>

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A., Ma, Y., Wong, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+effects+of+cancer+mitochondria+on+energy+metabolism+and+tumorigenesis:+utility+of+transmitochondrial+cybrids.">Functional effects of cancer mitochondria on energy metabolism and tumorigenesis: utility of transmitochondrial cybrids.</a> Ann N Y Acad Sci. 1201, 137-146 (2010).</li><li>Wu, T. 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=Mitochondrial+Transfer+by+Photothermal+Nanoblade+Restores+Metabolite+Profile+in+Mammalian+Cells.">Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells.</a> Cell Metab. 23 (5), 921-929 (2016).</li><li>Masuzawa, 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=Transplantation+of+autologously+derived+mitochondria+protects+the+heart+from+ischemia-reperfusion+injury.">Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury.</a> Am J Physiol Heart Circ Physiol. 304 (7), H966-H982 (2013).</li><li>Kitani, T., 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=Direct+human+mitochondrial+transfer:+a+novel+concept+based+on+the+endosymbiotic+theory.">Direct human mitochondrial transfer: a novel concept based on the endosymbiotic theory.</a> Transplant Proc. 46 (4), 1233-1236 (2014).</li><li>Caicedo, 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=MitoCeption+as+a+new+tool+to+assess+the+effects+of+mesenchymal+stem/stromal+cell+mitochondria+on+cancer+cell+metabolism+and+function.">MitoCeption as a new tool to assess the effects of mesenchymal stem/stromal cell mitochondria on cancer cell metabolism and function.</a> Sci Rep. 5, 9073 (2015).</li><li>Kesner, E. E., Saada-Reich, A., Lorberboum-Galski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Mitochondrial+Transformation+into+Human+Cells.">Characteristics of Mitochondrial Transformation into Human Cells.</a> Sci Rep. 6, 26057 (2016).</li><li>Lathia, J. D., Mack, S. C., Mulkearns-Hubert, E. E., Valentim, C. L., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+glioblastoma.">Cancer stem cells in glioblastoma.</a> Genes Dev. 29 (12), 1203-1217 (2015).</li><li>Osswald, 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=Brain+tumour+cells+interconnect+to+a+functional+and+resistant+network.">Brain tumour cells interconnect to a functional and resistant network.</a> Nature. 528 (7580), 93-98 (2015).</li><li>Shinojima, 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=TGF-beta+mediates+homing+of+bone+marrow-derived+human+mesenchymal+stem+cells+to+glioma+stem+cells.">TGF-beta mediates homing of bone marrow-derived human mesenchymal stem cells to glioma stem cells.</a> Cancer Res. 73 (7), 2333-2344 (2013).</li><li>Velpula, K. K., Dasari, V. R., Rao, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homing+of+human+cord+blood+stem+cells+to+sites+of+inflammation:+unfolding+mysteries+of+a+novel+therapeutic+paradigm+for+glioblastoma+multiforme.">The homing of human cord blood stem cells to sites of inflammation: unfolding mysteries of a novel therapeutic paradigm for glioblastoma multiforme.</a> Cell Cycle. 11 (12), 2303-2313 (2012).</li><li>Guichet, P. O., 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=Cell+death+and+neuronal+differentiation+of+glioblastoma+stem-like+cells+induced+by+neurogenic+transcription+factors.">Cell death and neuronal differentiation of glioblastoma stem-like cells induced by neurogenic transcription factors.</a> Glia. 61 (2), 225-239 (2013).</li><li>Lyons, E. A., Scheible, M. 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INSERM (Institut National de la Santé et de la Recherche Médicale), com a qual CJ ​​e ML.V. são filiados, entrou com um pedido de patente sobre a técnica MitoCeption (EP14306154.7).

Um número cada vez maior de estudos mostram que as células podem trocar mitocôndria e que estas mitocôndrias têm efeitos profundos sobre o metabolismo e as funções das células-alvo. Portanto, é essencial para dominar as ferramentas para transferir quantitativamente mitocôndrias das células do doador contra estas células alvo para permitir um estudo preciso dos seus efeitos biológicos. O protocolo aqui descrito foi originalmente funcionou para transferir mitocôndrias isoladas de...

As mitocôndrias são organelos encontrados em células eucarióticas em que desempenham um papel central na absorção de nutrientes, assim como na produção de energia e de metabolito. Essas organelas contêm DNA mitocondrial circular (mtDNA), 16,6 kb de comprimento, que codifica as proteínas dos complexos da cadeia de transporte de elétrons, tRNAs e rRNAs 1. A funcionalidade destes organelos é crítica para a homeostase celular e diversas patologias têm sido associados com a disfunção mitocondrial 1, 2, 3. O estado mitocôndria tem, por exemplo, sido associados a inflamação, doenças infecciosas e cancro, neste último caso, com consequências para a metástase e resistência à terapia com 4, 5, 6, 7. Mitocôndrias exibir a capacidade notável de<html> sendo transferidos entre &quot;doadores&quot; e células &quot;alvo&quot;. Isto leva a mudanças no metabolismo energético das células-alvo, bem como em outras modificações funcionais, tais como a reparação e resistência aos agentes quimioterapêuticos tecido, como foi recentemente mostrado por diferentes laboratórios 8, 9, 10, 11, 12, 13, 14, 15 , 16. As células estaminais mesenquimais humanas (MSCs) exibir esta capacidade de transferir mitocôndria para uma ampla variedade de células alvo, incluindo cardiomiócitos, células endoteliais, células epiteliais alveolares pulmonares, células tubulares renais e células cancerosas, conduzindo a alterações das propriedades funcionais destas células 8,&gt; 9, 10, 12, 17, 18. As mitocôndrias de troca aparece agora como um mecanismo amplamente utilizado que permite que um número de diferentes tipos de células para se comunicar uns com os outros e modificar as suas propriedades biológicas. Esta troca mitocôndrias pode ocorrer através da formação de túneis nanotubos (TNT), envolvendo conexina 43 contendo junções comunicantes 8 ou M-Sec / TNFaip2 eo exocyst complexos 19. Alternativamente, a transferência de mitocôndrias também mostrou ser mediada por arrestina microvesículas proteína contendo o domínio 1-mediadas (ARMMs 20). Curiosamente, a eficácia da transferência de mitocôndrias foi relacionado com a taxa de expressão da Rho GTPase 1 MIRO1 21, um factor chave para explicar as diferenças na mitocôndria eficácias de transferência entre iPSC-MSCs e adultos BM-MSCs 22. Apesar desta riqueza de dados relativos à troca de mitocôndria da célula-célula, relativamente pouco se sabe sobre a metabólico e resultados biológica dessa transferência mitocôndrias. Por isso, justifica plenamente a criação de instrumentos adequados para avaliar plenamente os efeitos biológicos desta transferência. Ao longo dos anos, várias abordagens técnicas para transferir mitocôndrias a partir de doadores de células aceitadores têm sido propostos. Isto inclui a injecção directa de mitocôndrias em oócitos de 23, 24, 25, fusão celular para gerar cybrids transmitochondrial 26, 27 e, mais recentemente, a transferência de mitocôndrias isoladas usando fototérmico nanolâminas 28. Nós e outros já demonstraram a capacidade de mitochond isoladoRia para ser internalizada pelas células vivas, como observado tanto in vitro como in vivo 29, 30, 31, através de mecanismos propostos para envolver macropinocitose 32. Desenvolvemos também um método, chamado MitoCeption, para transferir quantitativamente mitocôndrias isoladas (a partir de MSCs) às células alvo, tal como exemplificado com o (aderente) MDA-MB-231 linha celular de cancro da mama 31. Este protocolo foi adaptado aqui para a transferência de mitocôndrias isoladas de MSC humanas de glioblastoma células estaminais (GSCS). O glioblastoma é um tumor maligno agressivo do cérebro que rapidamente se tornam resistentes ao tratamento, principalmente devido às células estaminais do glioblastoma (GSC) presentes no interior do tumor 33. Estes GSCS crescer como neuroesferas in vitro e gerar tumores em modelos de xenoenxerto. As células cancerosas dentro de glioblastoma têm acapacidade para estabelecer ligações célula-para-célula, como demonstrado recentemente para as células tumorais do cérebro astrocíticos que interligam por meio de microtubos prolongadas, por meio da qual as mitocôndrias (bem como de cálcio e de células núcleos) pode migrar, resultando em redes astrocitoma radioterapia resistente 34. Glioblastoma pode recrutar muitas células diferentes dentro do microambiente do tumor, incluindo o MSC 35, 36. Nós mostramos que MSCs pode fazer conexões célula-célula com GSCS em co-cultura e transferir as suas mitocôndrias (dados não mostrados), que é esperado para modificar SGC propriedades funcionais. O presente protocolo descreve como a técnica MitoCeption pode ser usada para transferir as mitocôndrias, previamente isolado a partir de MSCs humanas, para GSCS humanos com a finalidade de se determinar o resultado biológico funcional. O multipotentes e altamente tumorigénicas linha GB4 SGC 37 foi utilizado neste estudo.

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	<tbody>
		<tr>
			<td>Mitochondria Isolation Kit for Tissue&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>10579663</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11520536</td>
		</tr>
		<tr>
			<td>B-27 Supplement W/O VIT A (50X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11500446</td>
		</tr>
		<tr>
			<td>HBSS w/o Ca2+ w/o Mg2+</td>
			<td>&#160;Sigma</td>
			<td>H4385</td>
		</tr>
		<tr>
			<td>poly Heme&#160;</td>
			<td>Sigma&#160;</td>
			<td>P3932</td>
		</tr>
		<tr>
			<td>aMEM w/o glutamine</td>
			<td>Ozyme</td>
			<td>BE12-169F</td>
		</tr>
		<tr>
			<td>DMEM/F-12 without glutamine,&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>11540566</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>25030-024&#160;</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma&#160;</td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>Insuline&#160;</td>
			<td>Sigma&#160;</td>
			<td>I 1882&#160;</td>
		</tr>
		<tr>
			<td>&#160;Human&#160;bFGF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
		</tr>
		<tr>
			<td>Human EGF&#160;</td>
			<td>Peprotech&#160;</td>
			<td>AF-100-15&#160;</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>MERCK</td>
			<td>2382</td>
		</tr>
		<tr>
			<td>Trypsine Inhibitor&#160;</td>
			<td>Sigma&#160;</td>
			<td>T9003</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>SIGMA&#160;</td>
			<td>10104159001</td>
		</tr>
		<tr>
			<td>Trypsine 0.25% /EDTA 1 mM</td>
			<td>Invitrogen&#160;</td>
			<td>25200056</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco&#160;</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>Protease inhibitors EDTA free</td>
			<td>Sigma</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>Ciprofloxacine&#160;</td>
			<td>Sigma</td>
			<td>17850-5G-F</td>
		</tr>
		<tr>
			<td>Fungine&#160;</td>
			<td>Invivogen</td>
			<td>ant-fn-1</td>
		</tr>
		<tr>
			<td>Fungizone&#160;</td>
			<td>Thermofisher</td>
			<td>15290018</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Euromedex</td>
			<td>EU0410</td>
		</tr>
		<tr>
			<td>&#160;MitoTracker Green FM</td>
			<td>Molecular Probes</td>
			<td>M7514</td>
		</tr>
		<tr>
			<td>&#160;MitoTracker Red CMXRos</td>
			<td>Molecular Probes</td>
			<td>&#160;M7512</td>
		</tr>
		<tr>
			<td>MitoTracker Deep Red FM</td>
			<td>Molecular Probes</td>
			<td>&#160;M22426&#160;</td>
		</tr>
		<tr>
			<td>&#160;CellTracker Green CMFDA</td>
			<td>Molecular Probes</td>
			<td>C7025</td>
		</tr>
		<tr>
			<td>&#160;CellTracker Blue CMF2HC</td>
			<td>Molecular Probes</td>
			<td>C12881</td>
		</tr>
		<tr>
			<td>RIPA</td>
			<td>Santa Cruz</td>
			<td>sc-24948</td>
		</tr>
		<tr>
			<td>FluoroDish Sterile Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Beckman Coulter</td>
			<td>2,523,749</td>
		</tr>
		<tr>
			<td>FACS apparatus</td>
			<td>Gallios&#160;</td>
			<td>&#160;3L 10C</td>
		</tr>
		<tr>
			<td>LC FAST START DNA MASTER PLUS&#160;</td>
			<td>Roche</td>
			<td>3515885001</td>
		</tr>
	</tbody>
</table>

mitoception: transferring isolated human msc mitochondria to glioblastoma stem cells

As mitocôndrias desempenham um papel central para o metabolismo celular, a produção de energia e de controlo de apoptose. função mitocondrial inadequada foi encontrado responsável por doenças muito diversas, que vão desde patologias neurológicas ao câncer. Curiosamente, as mitocôndrias foram recentemente mostrado para exibir a capacidade de ser transferidos entre os tipos de células, nomeadamente a partir de células estaminais mesenquimais humanas (MSC) para as células cancerosas em condições de co-cultura, com metabólicas e funcionais consequências para as células mitocôndrias destinatários, aumentando ainda mais o interesse actual para as propriedades biológicas destes organelos. Avaliação dos efeitos da mitocôndria MSC transferido nas células alvo é de importância primária para entender o resultado biológico de tais interacções célula-célula. O protocolo aqui descrito MitoCeption permite a transferência das mitocôndrias isoladas de antemão a partir das células dadoras para as células alvo, usando MSC mitocôndriase de células estaminais glioblastoma (GSC) como um sistema modelo. Este protocolo foi anteriormente usada para transferir as mitocôndrias, isolado a partir de MSC, para aderentes de células de cancro MDA-MB-231. Este protocolo de transferência de mitocôndrias é adaptado aqui para GSCS que apresentam a particularidade específica de crescer como neurospheres in vitro. A transferência das mitocôndrias isoladas pode ser seguido de células activadas por fluorescência (FACS) e de imagem confocal utilizando mitocôndrias corantes vitais. A utilização de doadores e células alvo as mitocôndrias, com diferentes haplótipos (SNPs), também permite a detecção das mitocôndrias transferidos com base na sua concentração de ADN mitocondrial circular (mtDNA) nas células alvo. Uma vez que o protocolo foi validado com estes critérios, as células que albergam as mitocôndrias transferidos pode ser ainda analisada para determinar os efeitos das mitocôndrias exógenos nas propriedades biológicas, tais como o metabolismo celular, plasticidade, proliferação e resposta à terapia.

Os passos processuais que descrevem o isolamento de mitocôndrias de células estaminais mesenquimais (MSC) e a sua transferência para as células-tronco glioblastoma segmentados (GSC) por MitoCeption são mostrados na Figura 1. GSCS são células-tronco cancerosas cultivadas como neurospheres para preservar suas propriedades de células-tronco. Para o protocolo, GSCS são semeadas como células individuais de um par de horas antes da transferência das mitocôndrias (p...

Watch this Scientific Journal Video about MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells at JoVE.com

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Aqui, um protocolo (MitoCeption) é apresentada para transferir mitocôndrias, isolado a partir de células estaminais mesenquimais humanas (MSC), para células estaminais glioblastoma (GSC), com o objectivo de estudar os seus efeitos biológicos sobre o metabolismo SGC e funções. Um protocolo semelhante pode ser adaptado para transferir as mitocôndrias entre outros tipos de células.

MitoCeption: Transferir humano isolado MSC mitocôndrias de células-tronco Glioblastoma

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

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Biology

12.7K visualizações.  Montpellier University.  Aqui, um protocolo (MitoCeption) é apresentada para transferir mitocôndrias, isolado a partir de células estaminais mesenquimais humanas (MSC), para células estaminais glioblastoma (GSC), com o objectivo de estudar os seus efeitos biológicos sobre o metabolismo SGC e funções. Um protocolo semelhante pode ser adaptado para transferir as mitocôndrias entre outros tipos de células. 

Vídeo: MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagação de-tronco embrionárias humanas (ES) Células

Biologia

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Contando células-tronco neurais

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Passaging células-tronco neurais

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

Imunocitoquímica: células-tronco neurais

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Cromatina imunoprecipitação de células estaminais embrionárias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificação de mitocôndrias de células de levedura

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelamento e descongelamento, células estaminais embrionárias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfectando e Nucleofecting células-tronco pluripotentes induzidas

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Parâmetro Multi-Medição da abertura dos poros transição de permeabilidade em mitocôndrias isoladas de coração Rato

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...