Agradecemos a Andrea Parmeggiani (L2C y DIMNP, Montpellier), Benoit Charlot (IES, Montpellier), así como los miembros del laboratorio útil para los debates, Christophe Duperray para ayudar con el análisis FACS, la instalación de imágenes RIO Montpellier (MRI) para proporcionar el medio ambiente adecuado para FACS y microscopía confocal. BNM fue apoyado por una beca de postgrado de la LABEX Numev (convención ANR-10-LabX-20). AB fue apoyado por una beca de estudiante de la Universidad de Varsovia y la Unión Europea (n ° POKL.04.01.02-00-221 / 12). MLV es un miembro del personal científico del Centro Nacional de Investigación Científica (CNRS).

Día 1 1. El etiquetado de la célula madre mesenquimales (MSC) Las mitocondrias (Opcional) <ol><li> Dos días antes de la preparación mitocondrias, semilla de MSC humanas en una placa de cultivo de 100 mm, en 10 ml aMEM / FBS 10%, a fin de tener 4 x 10 5 MSCs en cultivo en el Día 1. </li><li> Enjuagar las MSC con PBS (4 ml) y añadir 4 ml aMEM / FBS al 1% (precalentado a 37 ° C). </li><li> Añadir la cantidad requerida de las mitocondrias colorante vital e incubar las células durante 30 min en la incubadora a 37ºC. </li><li> Retire la solución de colorante mitocondrias, enjuagar las células dos veces con 4 ml precalentada (37 ° C) aMEM / FBS al 1% y volver a agregar 4 ml aMEM / FBS al 10%. Se incuban las células a 37 ° C. </li><li> Cambiar el medio de cultivo (10 ml aMEM / FBS 10%) después de 30 min y, otra vez, 2 horas más tarde. </li></ol> Día 1 2. El etiquetado de las células de glioblastoma madre (GSC) (Opcional, ver la sección Discusión) <table border="1" fo:keep-together.within-page = &quot;1&quot; fo: keep-con-next.within-page = &quot;always&quot;&gt; <tbody><tr><td> Medio de cultivo celular </td><td> Composición </td></tr><tr><td rowspan="5"> medio basal GSC </td><td> DMEM / F-12 suplementado con </td></tr><tr><td> La insulina 20 mg / ml </td></tr><tr><td> 1x suplemento N2 </td></tr><tr><td> La glucosa 3 g / L </td></tr><tr><td> L-glutamina 2 mM </td></tr><tr><td rowspan="9"> medio de proliferación SGC </td><td> Añadir al medio 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> Fungizone 0,25 mg / ml </td></tr><tr><td> Heparina 2 mg / ml </td></tr><tr><td> La ciprofloxacina 2 mg / ml </td></tr><tr><td> Gentamicina 2 mg / ml </td></tr><tr><td rowspan="4"> medio de proliferación MSC </td><td> &amp; #945; MEM suplementado con </td></tr><tr><td> L-glutamina 2 mM </td></tr><tr><td> 10% de FBS </td></tr><tr><td> bFGF 2 ng / ml </td></tr></tbody></table> Tabla 1: Medios de cultivo. <ol><li> Disociar GSCS (línea celular Gb4 32 (10 x 10 6 células) crecido como neuroesferas en poli-HEMA frascos de cultivo celular recubierto (consulte los pasos 3.1 a 3.12). </li><li> GSCS de semillas en una placa de 48 pocillos a 10 5 células / pocillo en medio de proliferación GSC (500 l) (ver Tabla 1). </li><li> Centrifugar la placa a 270 xg durante 5 minutos a 20 ° C. </li><li> Añadir la cantidad requerida de colorante vital de células y se incuba durante 30 min a 37 ° C. </li><li> Añadir 500 l de medio basal GSC (Tabla 1) por pocillo de la placa de 48 pocillos. </li><li> Centrifugar la placa a 270 xg, a 20 ° C durante 5 min. Aspirar el sobrenadante. </li><li> Repita los pasos 2.5 a 2.6. </li><li> Añadir 500 l de medio basal GSC y se incuba a 37 ° C durante 30 min. </li><li> Centrifugar la placa a 270 xg, a 20 ° C durante 5 min. Aspirar el sobrenadante. </li><li> Añadir 500 l de medio de proliferación GSC por pocillo de la placa de 48 pocillos y se incuban en la incubadora a 37ºC. Nota: La cantidad de GSCS indicado (10 x 10 6 células) permite la realización de los diferentes experimentos de dosis-respuesta y los controles de FACS. Una vez que las condiciones experimentales se definen con mayor precisión esta cantidad puede ser mucho menos marcada. </li></ol> Dia 2 3. La siembra de las células madre de glioblastoma <ol><li> Recoger las neuroesferas GSC (10 x 10 6 células) por centrifugación a 270 xg durante 5 min, a 20 ° C en un tubo de 50 ml. </li><li> Lavar las células con 5 ml de HBSS, y se centrifuga a 270 xg durante 5 min a 20 ° C. </li><li> Aspirar el sobrenadante. </li><li> resuspender suavemente el sedimento de GSC en 100 l de tripsina-EDTA (0,25%) (por 10 x 10 <sarriba&gt; 6 células) </li><li> Incubar a 37 ° C durante 3 min. </li><li> Añadir 10 l CaCl DNasa 2 (20 mM) y 2 l I (10 mg / ml). </li><li> Disociar las neuroesferas pipeteando suavemente hacia arriba y hacia abajo (30-50x) con una pipeta P200. Evitar las burbujas. Compruebe bajo el microscopio que todos GSCS se disocian. </li><li> Añadir 10 l inhibidor de tripsina (5%) y 10 ml de HBSS. </li><li> Centrifugar a 270 xg GSCS durante 7 minutos a 20 ° C. </li><li> Eliminar el sobrenadante y añadir 10 ml de medio basal GSC (Tabla 1). </li><li> Contar GSCS con una cámara de recuento Thoma y centrifugar las células a 270 xg durante 7 minutos a 20 ° C. </li><li> Añadir el volumen adecuado de medio de proliferación GSC (Tabla 1) para llegar a la concentración celular de 10 6 GSCS / ml. </li><li> De semillas 10 5 GSCs (100 l de la suspensión de células) por pocillo de una placa de 96 pocillos. </li><li> Centrifugar las placas a 270 x g durante 7 minutos a 20 ° C para obtener GSCS en el bottom de pozos. </li><li> Incubar la placa de 96 pocillos a 37 ° C hasta que la sección 5. </li></ol> Dia 2 4. Aislamiento de MSC mitocondrias <ol><li> Ajuste la temperatura del microtubo de centrifugación a 4 ° C. </li><li> Preparar dos tubos de 1,5 ml &quot;A&quot; y &quot;C&quot;, que contiene los reactivos para la extracción de las mitocondrias (200 l de reactivo A y 400 l de reactivo C, que contiene tanto los inhibidores de la proteasa libre de EDTA). Preparar otros 2 tubos, etiquetados &quot;MSC&quot; y &quot;Mito&quot;. Mantenga todos los tubos en hielo. También hay que preparar los tubos para las diluciones seriadas de las mitocondrias. </li><li> Wash MSCs con 10 ml precalentada (37 ° C) PBS. </li><li> Wash MSCs con 2 ml de tripsina (sin EDTA) durante 10 segundos y añadir 1 ml de tripsina (sin EDTA). Se incuban las células durante 5-10 minutos a 37 ° C. </li><li> Recuperar las MSC mediante la adición de 10 ml aMEM / FBS al 10%, la transferencia a un tubo de 50 ml. </li><li> centrifugar las células a 270 xg durante 5 minutos a 20 ° C. </li><li> Eliminar el sobrenadante y añadir10 ml aMEM / FBS 10% al sedimento celular. </li><li> Contar las MSCs con una cámara de recuento Malassez. </li><li> Centrifugar MSCs (4-5 x 10 5) a 270 xg durante 5 min a 20 ° C. </li><li> Descartar el sobrenadante, añadir 1 m enfriado en hielo aMEM / FBS 10% al sedimento celular y la transferencia de las células al tubo marcado con &quot;MSC&quot; (preparado en el paso 4.2). Mantenga el tubo en hielo. </li><li> Centrifugar el tubo que contiene las MSCs en 900 xg durante 5 min, a 4 ° C. </li><li> Eliminar todo el medio residual del tubo. </li><li> Añadir 200 l de mitocondrias Aislamiento Reactivo A (que contiene los inhibidores de la proteasa libre de EDTA). Vortex a velocidad media durante 5 seg y dejar los tubos en hielo durante exactamente 2 min. </li><li> Añadir 2,5 l de mitocondrias Aislamiento Reactivo B. Vortex a velocidad máxima durante 10 segundos, y luego se van tubos en hielo. Repetir cada 30 s durante 5 min. </li><li> Añadir 200 l de mitocondrias Aislamiento Reactivo C (que contiene los inhibidores de proteasa libre de EDTA). Mezclar por la inclinación del tubo (roughly 30 veces, no vórtice). Centrifugar el tubo a 700 xg durante 10 min a 4 ° C. </li><li> Transferir el sobrenadante (que contiene la mitocondria MSC) al tubo &quot;Mito&quot; (preparado en el paso 4.2). </li><li> Centrifugar a 3000 xg, 15 min a 4 ° C, para obtener el pellet mitocondrias. Descartar el sobrenadante. El sedimento contiene las mitocondrias aisladas. </li><li> Enjuague el pellet mitocondrias con 200 l de reactivo C. A continuación, se centrifuga a 12.000 xg, 5 min a 4 ° C, para obtener el pellet mitocondrias. </li></ol> 5. Transferencia de mitocondrias aisladas MSC a GSCS (MitoCeption) <ol><li> Añadir 200 l de la (0 ° C) medio pre-enfriado GSC proliferación al sedimento de mitocondrias aisladas de MSCs (4 x 10 5). </li><li> Se diluye la preparación de las mitocondrias (en medio de proliferación SGC) para añadir consistentemente 20 l de suspensión mitocondrial de los GSCS. </li><li> Añadir el volumen de mitocondrias aisladas en los pocillos de la 96-wplaca ell que contiene los GSCs (de la etapa 3.15), a la concentración deseada (de 0,1 a 10 mg). Añadir la mitocondria lentamente, cerca de la parte inferior del pozo, que cubre toda la superficie al menos una vez. </li><li> Para el control de MSC mitocondrias fuga de colorante vital, añadir la misma cantidad de la preparación mitocondrias (0,1 a 10 mg) a los pocillos de una placa de 96 pocillos que contenía GSC medio de proliferación solamente (sin GSCS) (véase la parte 6.2 para la detección de fugas de colorante vital). </li><li> Centrifugar la placa de 96 pocillos que contiene las mitocondrias GSCs receptores con la mitocondria MSC (etapa 5.3) y la placa de control (paso 5.4) a 1.500 xg durante 15 min a 4 ° C. </li><li> Colocar las placas de cultivo en el 37 ° C incubadora de células inmediatamente después de la centrifugación. Nota: El protocolo de transferencia de las mitocondrias se basa en la centrifugación de la suspensión mitocondrias en las células cultivadas en la fuerza de centrifugación adecuada, con una serie de centrifugaciones que se puede ajustar en función de la System células de donante / receptor mitocondrias. </li></ol> Día 3 6. Análisis de la transferencia de mitocondrias por FACS y Confocal Imaging <ol><li> Preparación de muestras para análisis FACS GSC Nota: Si las mitocondrias MSC se marcaron con un colorante vital de antemano (Sección 1), la eficiencia puede ser monitoreado por FACS, 24 horas después de la transferencia de las mitocondrias. <ol><li> Centrifugar la placa de 96 pocillos con los GSCs MitoCepted a 270 xg durante 5 min a 20 ° C. </li><li> Descartar el sobrenadante. </li><li> Añadir 100 l de tripsina (sin EDTA) en cada pocillo. Incubar a 37 ° C durante 3 min. Pipetear hacia arriba y hacia abajo para disociar las neuroesferas formadas en el periodo de tiempo de 24 horas. </li><li> Añadir 100 l de medio basal SGC. </li><li> Centrifugar la placa a 270 xg durante 5 min a 20 ° C y descartar el sobrenadante. </li><li> Resuspender GSCS en 300 l GSC medio basal y la transferencia de FACS adaptado tubos. </li><li> Realice la una FACSANÁLISIS. </li></ol></li><li> El control de fugas mitocondrias colorante vital de las mitocondrias aisladas (FACS) Nota: El propósito de este paso es determinar la señal de FACS de fondo que no reflejaría una verdadera transferencia MSC mitocondrias sino, más bien, la mera fuga de colorante vital de la mitocondria MSC marcado utilizado para MitoCeption (paso 5.3). <ol><li> Sembrar las células GSC como se describe en los pasos 3.1 a 3.15. </li><li> Se incuban las células a 37 ° C durante 1 hr. </li><li> Centrifugar la placa de 96 pocillos que contiene mitocondrias solamente (de la etapa 5.4 a 5.6) a 1500 xg durante 5 min a 20 ° C. </li><li> Aspirar el medio de cultivo de la placa de 96 pocillos GSC (paso 6.2.2) y sustituirlo por el medio incubado con mitocondrias solamente (sobrenadante del paso 6.2.3). </li><li> Incubar la placa de 96 pocillos que contiene los GSCs a 37 ° C durante 2 hr. </li><li> Proceder como en los pasos 6.1.1 a 6.1.7 para el análisis FACS. </li></ol></li><li> Preparación de GSC samples de imagen confocal Nota: Si la transferencia de MSC mitocondrias para GSCS va a ser analizada por imágenes de fluorescencia, ambos MSC y GSCS necesitan ser etiquetados con anterioridad, respectivamente, con las mitocondrias (paso 1) y celular (paso 2) colorantes vitales. <ol><li> Preparar GSCS como en los pasos 6.1.1 a 6.1.5. </li><li> Volver a suspender en 2 ml GSCS GSC medio de proliferación que contenía FBS al 2% y las semillas en placas de cultivo de 35 mm (fondo de cristal). </li><li> Realizar imagen confocal en las GSCS con las mitocondrias fluorescentes MSC transferidos 24 horas más 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. K., Sturk-Andreaggi, K., Irwin, J. A., Just, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+Sanger+strategy+for+human+mitochondrial+genome+sequencing.">A high-throughput Sanger strategy for human mitochondrial genome sequencing.</a> BMC Genomics. 14, 881 (2013).</li></ol>

INSERM (Institut National de la Santé et de la Recherche Médicale), con el que CJ y ML.V. están afiliados, ha presentado una solicitud de patente de la técnica MitoCeption (EP14306154.7).

Un creciente número de estudios muestran que las células pueden intercambiar las mitocondrias y que estas mitocondrias tener profundos efectos en el metabolismo y las funciones de la célula diana. Por lo tanto, es esencial para dominar las herramientas para transferir cuantitativamente la mitocondria de las células del donante a estas células diana para permitir un estudio preciso de sus efectos biológicos. El protocolo descrito aquí fue originalmente funcionó para transferir las mit...

Las mitocondrias son orgánulos que se encuentran en las células eucariotas en los que juegan un papel central en la absorción de nutrientes, así como en la producción de energía y metabolitos. Estos orgánulos contienen ADN mitocondrial circular (mtDNA), 16,6 kb de largo, que codifica las proteínas de los complejos de la cadena de transporte de electrones, tRNAs y rRNAs 1. La funcionalidad de estos orgánulos es crítica para la homeostasis celular y varias patologías se han asociado con disfunción mitocondrias 1, 2, 3. El estado de las mitocondrias, por ejemplo, ha sido relacionado con la inflamación, las enfermedades infecciosas y el cáncer, en este último caso con consecuencias para la metástasis y la resistencia a la terapia de 4, 5, 6, 7. Las mitocondrias muestran la notable capacidad de las<html> siendo transferido entre las células &quot;diana&quot; &quot;donante&quot; y. Esto conduce a cambios en el metabolismo energético de las células diana, así como en otras modificaciones funcionales, tales como la reparación de tejidos y la resistencia a los agentes quimioterapéuticos, como demostrado recientemente por diferentes laboratorios 8, 9, 10, 11, 12, 13, 14, 15 , 16. Las células madre mesenquimales humanas (MSCs) muestran esta capacidad de transferencia de las mitocondrias a una amplia variedad de células diana, incluyendo cardiomiocitos, células endoteliales, células epiteliales alveolares pulmonares, células tubulares renales y las células cancerosas, lo que lleva a las modificaciones de las propiedades funcionales de estas células 8,&gt; 9, 10, 12, 17, 18. Las mitocondrias de cambio aparece ahora como un mecanismo ampliamente utilizado que permite que un número de diferentes tipos de células para comunicarse unos con otros y modificar sus propiedades biológicas. Este intercambio mitocondria puede ocurrir a través de la formación de los nanotubos de efecto túnel (TNT), con la participación de conexina 43 que contienen uniones gap 8 o M-Sec / TNFaip2 y la exocyst complejas 19. Alternativamente, la transferencia de las mitocondrias también ha demostrado ser mediada por arrestina microvesículas proteína que contiene el dominio 1 (mediadas por ARMMs) 20. Curiosamente, la eficacia de la transferencia de las mitocondrias estaba vinculada a la tasa de expresión de la Rho GTPasa 1 miro1 21, un factor clave para explicar las diferencias en las eficacias de transferencia entre las mitocondrias iPSC-MSC y adultos BM-MSC 22. A pesar de esta gran cantidad de datos relativos a las mitocondrias de cambio de célula a célula, se sabe relativamente poco sobre el metabolismo y la evolución biológica de esta transferencia de las mitocondrias. Por lo tanto, completamente de acuerdo con la configuración de las herramientas apropiadas para evaluar plenamente los efectos biológicos de esta transferencia. A través de los años, varios enfoques técnicos para la transferencia de las mitocondrias de donante a las células aceptoras se han propuesto. Esto incluye la inyección directa de las mitocondrias en oocitos de 23, 24, 25, la fusión celular para generar cíbridos transmitochondrial 26, 27 y, más recientemente, la transferencia de mitocondrias aisladas utilizando fototérmica nanoblades 28. Nosotros y otros previamente demostrada la capacidad de mitochond aisladoria para ser internalizado por las células vivas, como se observa tanto in vitro como in vivo 29, 30, 31, a través de los mecanismos propuestos para involucrar a macropinocitosis 32. Hemos desarrollado además un método, llamado MitoCeption, para transferir cuantitativamente mitocondrias aisladas (de MSCs) a las células diana, como se ejemplifica con el (adherente) MDA-MB-231 línea celular de cáncer de mama 31. Este protocolo fue adaptado aquí para la transferencia de las mitocondrias aisladas MSC humanos a las células madre de glioblastoma (GSCS). Glioblastoma son tumores malignos agresivos del cerebro que rápidamente se vuelven resistentes al tratamiento, debido principalmente a las células madre glioblastoma (GSC) presentes dentro del tumor 33. Estos GSCs crecen como neuroesferas en vitro y generan tumores en modelos de xenoinjerto. Las células cancerosas dentro de glioblastoma tienen lacapacidad para hacer conexiones de célula a célula, como se muestra recientemente para células tumorales cerebrales astrocíticos que se interconectan a través de microtubos extendidos, a través del cual las mitocondrias (así como núcleos de calcio y de células) pueden migrar, lo que resulta en las redes de astrocitoma de radioterapia resistente 34. Glioblastoma puede reclutar muchas células diferentes dentro del microambiente del tumor, incluyendo MSCs 35, 36. Nos mostró que las MSC puede realizar conexiones entre células con GSCS en co-cultivo y transferir sus mitocondrias (datos no mostrados), que se espera para modificar las propiedades funcionales de la SGC. El presente protocolo describe cómo la técnica MitoCeption se puede utilizar para transferir las mitocondrias, de antemano aislada de MSC humanas, a GSCs humanos con el propósito de determinar su resultado biológico funcional. El multipotentes y altamente tumorigénico línea Gb4 GSC 37 se utilizó en este estudio.

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		<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>
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			<td>Glucose&#160;</td>
			<td>Sigma&#160;</td>
			<td>G7021</td>
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		<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

Las mitocondrias juegan un papel central para el metabolismo celular, la producción de energía y el control de la apoptosis. la función mitocondrial inadecuada ha sido encontrado responsable de muy diversas enfermedades, que van desde patologías neurológicas al cáncer. Curiosamente, las mitocondrias recientemente se ha demostrado para mostrar la capacidad de ser transferido entre los tipos de células, en particular a partir de células madre mesenquimatosas humanas (MSC) a las células cancerosas en condiciones de cocultivo, con metabólicos y funcionales consecuencias para las células mitocondrias receptores, mejorando aún más el interés actual para las propiedades biológicas de estos orgánulos. La evaluación de los efectos de la mitocondria MSC transferido en las células diana es de primordial importancia para entender el resultado biológico de tales interacciones célula-célula. El protocolo MitoCeption descrito aquí permite la transferencia de las mitocondrias aisladas de antemano a partir de las células del donante a las células diana, usando MSC mitocondriasy células madre de glioblastoma (GSC) como un sistema modelo. Este protocolo se ha utilizado anteriormente para transferir las mitocondrias, aislado de MSCs, a adherente células cancerosas MDA-MB-231. Este protocolo de transferencia de las mitocondrias está adaptado aquí para GSCS que presentan la particularidad específica de crecer como neuroesferas in vitro. La transferencia de las mitocondrias aisladas puede ir seguida de células activadas por fluorescencia (FACS) y la imagen confocal utilizando mitocondrias colorantes vitales. El uso de células donantes y de destino mitocondrias con haplotipos distintos (SNP) también permite la detección de la mitocondria transferidos sobre la base de la concentración de su ADN mitocondrial circular (ADNmt) en las células diana. Una vez que el protocolo ha sido validado con estos criterios, las células que albergan la mitocondria transferidos se pueden analizar adicionalmente para determinar los efectos de las mitocondrias exógenos en las propiedades biológicas, tales como el metabolismo celular, la plasticidad, la proliferación y respuesta al tratamiento.

Los pasos de procedimiento que describen el aislamiento de las mitocondrias de las células madre mesenquimales (MSC) y su transferencia a las células madre glioblastoma dirigidos (GSC) por MitoCeption se muestran en la Figura 1. GSCS son las células madre cancerosas cultivadas como neuroesferas para preservar sus propiedades de células madre. Para el protocolo, GSCS se siembran como células individuales de un par de horas antes de la transferencia de las mitocondria...

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

Aquí, un protocolo (MitoCeption) se presenta para transferir las mitocondrias, aislado a partir de células madre mesenquimatosas humanas (MSC), a las células madre glioblastoma (GSC), con el objetivo de estudiar sus efectos biológicos sobre el metabolismo y funciones GSC. Un protocolo similar se puede adaptar para transferir mitocondrias entre otros tipos de células.

MitoCeption: Transferencia hermoso humano MSC mitocondrias de las células madre de 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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JoVE Journal

Biology

12.7K vistas.  Montpellier University.  Aquí, un protocolo (MitoCeption) se presenta para transferir las mitocondrias, aislado a partir de células madre mesenquimatosas humanas (MSC), a las células madre glioblastoma (GSC), con el objetivo de estudiar sus efectos biológicos sobre el metabolismo y funciones GSC. Un protocolo similar se puede adaptar para transferir mitocondrias entre otros tipos de células. 

Video: 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.

Propagación de la madre embrionarias humanas (ES) Células

Investigación

Biología

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.

Conteo de células madre neurales humanas

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.

Pases células madre neurales humanas

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.

Inmunocitoquímica: células madre neurales humanas

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.

Inmunoprecipitación de la cromatina de células estaminales embrionarias 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.

Purificación de las mitocondrias de las células de levadura

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.

Congelación y descongelación células estaminales embrionarias 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.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

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.

Transfección y Nucleofecting Humanos células madre pluripotentes inducidas

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.

Multi-parámetro de medición de la apertura del poro de transición de permeabilidad en el corazón del ratón mitocondrias aisladas

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