Trabajo que se describe en este manuscrito fue posible gracias a la financiación del Instituto de Medicina Regenerativa de California (CIRM) para Core UCR de células madre.

Nuestro protocolo se inicia con un método de adaptación CMPi humanos para liberar a las culturas de alimentación, seguido por los protocolos para la transfección CMPi humanos utilizando GeneJuice (EMD) y nucleofection de CMPi humano utilizando un dispositivo nuclefector Amaxa. Nota: Los siguientes procedimientos se llevan a cabo en una campana de flujo laminar estéril. Todos los medios de comunicación y soluciones equilibradas a 37 ° C de temperatura ambiente antes de comenzar o menos que se especifique lo contrario. 1. El establecimiento de CMPi humanos en el alimentador sin sistema CMPi humanos previamente mantenido en las células de alimentación se puede dividir, transferido a Geltrex recubiertos plato y se mantiene durante dos pasajes antes de la alimentación libre de transfección. <ol><li> Descongele Geltrex la noche a 4 ° C. Para preparar el recubrimiento Geltrex, diluir descongelado Geltrex 1:50 en DMEM frío. Mezclar suavemente las soluciones. </li></ol> Nota: Geltrex, como Matrigel es una forma soluble de la matriz de la membrana basal purificada a partir de células murinas Engelbreth-Holm-Swarm tumor (EHS). Por otra parte, Matrigel se puede utilizar como una matriz extracelular para establecer alimentador de cultivos libres de iPSC humanas. <ol start="2"><li> Cubrir toda la superficie de los pozos de la cultura con Geltrex solución (1 ml de una de 35 mm también). Escudo pozos con Geltrex a 37 ° C incubadora durante 1 hora. </li><li> Para CMPi paso humano, añadir 1 ml de accutase por pocillo y se incuba a 37 ° C durante 1 minuto hasta que la mayoría de las células comienzan a separarse. </li></ol> Para CMPi paso humano, añadir 1 ml de accutase por pocillo y se incuba a 37 ° C durante 1 minuto hasta que la mayoría de las células comienzan a separarse. <ol start="4"><li> Añadir 10-15 perlas de vidrio a las células y agite suavemente la placa. Añadir 2 ml de golpe de gracia DMEM / F12 y triturar con suavidad. Transferencia de suspensión de células a un tubo de centrífuga de 10 ml. </li><li> Girar las células a 800 rpm durante 3 min a temperatura ambiente. </li><li> Aspirar el sobrenadante del tubo, dejando humanos pellet iPSC intacto. Agitar suavemente el tubo para dispersar sedimento celular. </li><li> Quitar Geltrex del pozo cubierto. </li><li> Resuspenda suavemente humanos pellet de IPSC en un volumen adecuado de STEMPRO. Distribuir entre los pozos de los alimentadores, en función de la tasa de proliferación). CMPi humano puede pasarse de una relación de división de 1:2 a 1:6. </li><li> Con cuidado, coloque en 5% de CO 2 incubadora, agitar la placa con cuidado para asegurar una distribución uniforme de las células a través de los pozos. </li><li> Alimentación de las celdas al día hasta que las células están listos para ser dividido de nuevo (cuando las células alcanzan el 80% de confluencia). </li><li> Paso a nuevas humanos CMPi Geltrex recubierto así (paso 1.3 a 1.9) en una relación de división de 1:2. Pequeñas colonias deben ser formados y distribuidos de manera uniforme en Geltrex recubiertos bien antes de la transfección. </li></ol> 2. Transfección de CMPi humanos con GeneJuice Las células (cultivadas en placas de 6 pocillos) debe ser de aproximadamente 40 a 50% confluentes en el día de la transfección de lograr la eficacia de transfección óptima. No es necesario cambiar el medio celular hasta el día siguiente. <ol><li> Preparar 100 KO-DMEM/F12 l en un tubo estéril eppendorf de 1,5 ml. Añadir 27 l de reactivo de transfección GeneJuice. Mezclar bien. Incubar a temperatura ambiente durante 5 minutos. </li><li> Añadir 4 g de ADN plásmido. Incubar el tubo a temperatura ambiente durante 15 minutos. La elección de los plásmidos es fundamental para la óptima eficiencia de transfección. Nosotros usamos un plásmido con una proteína de fluorescencia verde mayor (eGFP) impulsado por el promotor CAG 5 (pCAG-eGFP). CAG promotor es un promotor fuerte que es transcripcionalmente activo en CMPi humanos y por lo tanto se puede utilizar para conducir la expresión del transgén en las células. En nuestras manos, linearización del plásmido no parece afectar a la eficacia de transfección. </li><li> Añadir GeneJuice mezcla de ADN a las células y agitar la placa. Haga girar la placa a 1200 rpm durante 5 minutos (método &quot;spinoculation &#39;) para aumentar el contacto de la mezcla de transfección con CMPi humanos en el pozo. </li><li> Se incuban las células a 37 ° C durante la noche. </li><li> Monitorear la eficiencia de transfección al día siguiente. </li><li> Para la transfección estable, el cambio medio al día siguiente con nuevas STEMPRO. Añadir la selección adecuada de antibióticos después de 24 a 48 horas. </li></ol> 3. Nucleofection de CMPi humanos CMPi humanos que han crecido en los alimentadores o Geltrex se puede utilizar para nucleofection. Sin embargo, se recomienda replating nucleofected CMPi humanos en fibroblastos de embriones de ratón (MEF) o fibroblastos de prepucio humano (HFF) alimentadores para garantizar la viabilidad celular alta y la recuperación. Por lo menos 2 millones de células se debe utilizar para lograr una mayor supervivencia celular tras nucleofection. <ol><li> Prepare la célula de enlace de medios condicionados (CM) por el golpe de gracia iPSC incubando suero humano de reemplazo (KOSR) los medios de comunicación con las células de alimentación durante la noche. CM recoger cada 24 horas. </li></ol> Nota: Cualquier cepa de ratón utilizada para la preparación de las capas de alimentación (como BLK6, CF1 y MF1) es adecuado para ser utilizado parar lo que CM. <ol start="2"><li> En el día de nucleofection, pre-tratamiento CMPi humanos con 10 inhibidor de rock M durante al menos 1 hora. </li><li> Preparar 82 l de solución de nucleofector humanos con células madre en un tubo estéril eppendorf de 1,5 ml. Añadir 18 l suplemento 1. Mezclar bien. Incubar la solución a 37 ° C durante 5 minutos. </li><li> Pre-caliente de alimentación de células acondicionado medios de comunicación (CM) y el 0,25% de tripsina / EDTA a 37 º C. Bajo el capó, prelabel una estéril tubo cónico de 15 ml. </li><li> Retire con cuidado los medios de comunicación de la cultura humana que se iPSC nucleofected. Lave suavemente las células con 2 ml de solución de fosfato buffer 1X (PBS) por pocillo. Aspirar el PBS. Añadir 1 ml de tripsina 0,25% por pozo. Se incuban las células a 37 ° C durante 3 minutos. </li><li> Triturar suavemente las células con 1000 ml punta de la pipeta. Lave la parte inferior del pozo para asegurarse de que todos los CMPi humanos son completamente separados. Transferencia de suspensión de células en el tubo de 15 ml etiquetados cónica. </li></ol> Nota: tripsina en células individuales deben ser estrictamente evitados. Las células sólo se debe desplazarse en pequeños grupos de células contaba con aproximadamente trillizos de las células. Pequeños grupos de células (células triples) se disocia en células individuales durante la posterior manipulación de las células. <ol start="7"><li> Añadir 9 ml MEF medios para inactivar la tripsina. Girar las células a 800 rpm durante 3 minutos. Con cuidado aspirar el sobrenadante y dejar el pellet de células intactas. </li><li> Resuspender las células en precalentado 100 l de solución de nucleofector humanos con células madre a partir del paso 3.3. </li><li> La transferencia de las células a una cubeta nucleofector con un 1 ml punta de la pipeta. </li><li> Añadir 4 g de ADN plásmido en la suspensión celular en la cubeta. Las células y el ADN mezcla agitando lentamente. Toque en la cubeta dos veces en la superficie de la campana. </li><li> Insertar la cubeta en el soporte nucleofector. Utilice los programas de B-016. Células Nucleofect pulsando el botón X. </li></ol> &lt; <ok> &gt; Se mostrará cuando el proceso de nucleofection se ha completado (por lo general sólo tiene 5.1 segundos). El uso de los programas de nucleofection otros (A-023, A-033 y U-023) produjo menos del 10% de células positivas eGFP de CMPi humanos. </ok><ol start="12"><li> Recuperar las células nucleofected de la cubeta utilizando para ello la pipeta Pasteur de plástico. Recuperar las células de la resuspensión en CM precalentado y el inhibidor de piedra en un tubo estéril eppendorf de 1,5 ml. Las células se incuban a 37 ° C durante 10 minutos para permitir que las células se recuperan. </li><li> Transferencia de sabios de abandono en las capas de alimentación utilizando una punta de la pipeta ml células. Se incuban las células a 37 ° C durante la noche. </li><li> Monitorear la eficiencia de transfección al día siguiente. Para obtener clones estables de transgénicos CMPi humanos que expresan establemente transgén, el cambio medio al día siguiente con CM y el inhibidor de ROCK. Añadir la selección adecuada de antibióticos después de 24 a 48 horas. </li></ol> 4. Los resultados representativos: <img alt="Figura 1" src="/files/ftp_upload/3110/3110fig1.jpg" /> Figura 1. Microfotografías de Riv1 CMPi humanos transfectadas con pCAG-eGFP. (A) eGFP que expresan las células transfectadas con Riv1 GeneJuice en Geltrex 12 horas después de la transfección. Las colonias de Riv1 CMPi humanos plateado y formado en los alimentadores siguientes nucleofection utilizando B-016 (B) y A-023 (C) del programa. (D) de forma estable eGFP que expresan humanos colonias iPSC con expresión eGFP omnipresente derivados de GeneJuice. (E) establemente transfectadas Riv1 CMPi humanos retenidos eGFP expresión constitutiva en la diferenciación del cuerpo embrioides.

<ol>
	<li>Liew, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells:+possibilities+for+human+cell+transplantation.">Human embryonic stem cells: possibilities for human cell transplantation.</a> Ann Med. 37, 521-532 (2005).</li><li>Eiges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Zwaka, T. P., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogous+recombination+in+human+embryonic+stem+cells.">Homogous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21, 319-321 (2003).</li><li>Siemen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Liew, C. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Xia, X., Ayala, M., Thiede, B. R., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-+and+in+vivo-induced+transgene+expression+in+human+embryonic+stem+cells+and+derivatives.">In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.</a> Stem Cells. 26, 525-533 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+culture+of+human+embryonic+stem+cells+in+conditioned+medium+for+efficient+genetic+modification.">Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.</a> Nat Protoc. 3, 1435-1443 (2008).</li><li>Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+mediates+high-efficiency+stable+gene+knockdown+and+transgene+expression+in+human+embryonic+stem+cells.">Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.</a> Stem Cells. 26, 1436-1443 (2008).</li><li>Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenesis+in+human+embryonic+stem+cells+as+a+novel+tool+to+define+the+human+neural+lineage.">BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.</a> Stem Cells. 27, 521-532 (2009).</li></ol>

No hay conflictos de interés declarado.

Nuestros protocolos de resultado en las técnicas de simple, robusto y altamente reproducible para introducir transgenes en CMPi humanos sin efectos tóxicos importantes y la muerte celular. CMPi humanos deben ser pasados ​​en pequeños grupos de células (células 5-10) y se colocaron en Geltrex en alta densidad (1:2) para asegurar la eficacia de transfección óptima en numerosas colonias pequeñas. De líneas de iPSC que son más propensos a la muerte celular y la diferenciación, mayor número de CMPi humanos (hasta 4 X 10 6 células) debe ser utilizado para un experimento de nucleofection sola. Ensayo de transfección transitoria genera un gran número de transgenes que expresan CMPi humanos dentro de un día. Transfectadas establemente clones iPSC suelen aparecer dentro de 7 días, y estas colonias transgénicos deben estar listos para ser recogidos dentro de tres semanas. El uso del promotor CAG se describe aquí se asegura la expresión en todas partes del reportero eGFP. En virtud de estas mejoras, los protocolos pueden ser alimentados en otras aplicaciones, incluyendo la sobreexpresión de inducción condicional, la derivación de líneas reportero linaje específico, caída del shRNA o siRNA, la orientación de genes y la recombinación homóloga.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> 0,25% de tripsina con EDTA </td><td> Invitrogen </td><td> 25200056 </td><td></td></tr><tr><td> 2-mercaptoetanol </td><td> Invitrogen </td><td> 21985-023 </td><td></td></tr><tr><td> 3 perlas de vidrio mm </td><td> Fisher Scientific </td><td> 11-312A </td><td> Cuentas de vidrio debe ser lavado con ácido clorhídrico (HCl) durante la noche, se enjuaga con hidróxido de sodio (NaOH) y agua destilada, y esterilizado por autoclave antes de su uso. </td></tr><tr><td> Celular Accutase disociación reactivo </td><td> Invitrogen </td><td> A11105-01 </td><td></td></tr><tr><td> Fibroblastos factor de crecimiento básico (bFGF) </td><td> Invitrogen </td><td> PHG0263 </td><td></td></tr><tr><td> DMEM (glucosa alta) </td><td> Lonza </td><td> 12-741 F </td><td></td></tr><tr><td> Suero fetal bovino </td><td> Invitrogen </td><td> 16000044 </td><td></td></tr><tr><td> Geltrex </td><td> Invitrogen </td><td> 12760013 </td><td> Factor de reducción del crecimiento </td></tr><tr><td> GeneJuice reactivo de transfección </td><td> EMD Biosciences </td><td> 70967 </td><td></td></tr><tr><td> Glutamax-I (100 veces) </td><td> Invitrogen </td><td> 35050061 </td><td> L-glutamina (Invitrogen, 25030081) también se puede utilizar en su lugar. </td></tr><tr><td> Humanos iPSC KOSR los medios de comunicación </td><td></td><td></td><td> 500 ml embrionarias humanas los medios de comunicación se compone de 390 ml golpe de gracia DMEM / F12, 100 ml de suero Replacer golpe de gracia, 5 ml Glutamax-I (100X), 5 ml AANE (100X), 500 l 2-mercaptoetanol (55 mm) y suplementado con 10 ng / ml de bFGF . </td></tr><tr><td> Golpe de gracia DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td></td></tr><tr><td> Golpe de gracia en suero de repuesto (KOSR) </td><td> Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> Los medios de comunicación fibroblastos de embriones de ratón </td><td></td><td></td><td> MEF medios de comunicación se compone de 90% FBS, AANE 1X, 1X Glutamax I-1X y piruvato de sodio en DMEM (glucosa alta) </td></tr><tr><td> Aminoácido no esencial, AANE? (100X) </td><td> Invitrogen </td><td> 11140050 </td><td></td></tr><tr><td> De células madre humanas nucleofector solución de un suplemento con </td><td> Lonza </td><td> VAPH-5012 </td><td></td></tr><tr><td> Tampón fosfato salino sin Ca 2 + y Mg 2 + </td><td> Invitrogen </td><td> 10010023 </td><td></td></tr><tr><td> Piruvato de sodio (100 veces) </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> STEMPRO media kit </td><td> Invitrogen </td><td> A1000701 </td><td> 500 ml STEMPRO los medios de comunicación se compone de 454 ml golpe de gracia DMEM / F12, 10 ml de suero STEMPRO sin suplemento de crecimiento (50 veces), 5 ml Glutamax-I (100X), 5 ml AANE (100X), 36 ml de BSA (albúmina de suero bovino, 25%) , 909 l 2-mercaptoetanol (55 mm) y suplementado con 10 ng / ml de bFGF. </td></tr></tbody></table>

transfecting and nucleofecting human induced pluripotent stem cells

La modificación genética es continuar siendo una herramienta esencial en el estudio de la biología de células madre y en el planteamiento de las posibles aplicaciones clínicas de las células madre embrionarias humanas (células madre embrionarias humanas) 1. Mientras que las mejoras en varios métodos de entrega de genes se han descrito 2.9, sigue siendo un proceso de transfección de células madre embrionarias humanas caprichosa, y aún no ha sido reportado en humanos células madre pluripotentes inducidas (CMPi). En este video, que demuestran cómo nuestro laboratorio de rutina y transfecta nucleofects CMPi humanos utilizando el plásmido con un aumento de proteína verde fluorescente (eGFP) periodista. CMPi humanos se adaptan y se mantiene como alimentador de cultivos libres para eliminar la posibilidad de la transfección de células de alimentación y para permitir la selección eficiente de los clones transgénicos estable iPSC después de transfección. Para nucleofection, CMPi humanos son pre-tratados con inhibidores de rock 11 de tripsina en pequeños grupos de células, y nucleofected replated en los alimentadores en el alimentador de la célula-medio condicionado para mejorar la recuperación celular. Transgenes que expresan CMPi humanos se pueden obtener después de 6 horas. Selección del antibiótico se aplica después de 24 horas y de líneas transgénicas estables aparecen dentro de una semana. El protocolo es robusto y reproducible de líneas de iPSC sin alterar la pluripotencia de estas células.

Watch this Scientific Journal Video about Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells at JoVE.com

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

A pesar de los recientes avances en la modificación genética, la transfección de células madre embrionarias humanas (células madre embrionarias humanas) sigue siendo un proceso caprichoso. Para nuestro conocimiento, los métodos sistemático y eficiente para transfectar las células madre pluripotentes inducidas (CMPi) no se han reportado. Aquí se describen los protocolos robustos para transfectar eficiente y nucleofect CMPi humanos.

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 ...

transfecting-and-nucleofecting-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

21.3K vistas.  University of California Riverside. A pesar de los recientes avances en la modificación genética, la transfección de células madre embrionarias humanas (células madre embrionarias humanas) sigue siendo un proceso caprichoso. Para nuestro conocimiento, los métodos sistemático y eficiente para transfectar las células madre pluripotentes inducidas (CMPi) no se han reportado. Aquí se describen los protocolos robustos para transfectar eficiente y nucleofect CMPi humanos.

Video: Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfección de células madre neurales humanas con el Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Investigación

Biología

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Generación de células madre pluripotentes inducidas por reprogramación de fibroblastos humanos con los Lentivirus Stemgent Humanos TF Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Selección y aislamiento de las colonias de humanos células madre pluripotentes inducidas a partir de fibroblastos adultos Reprogramado

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

La reprogramación de células somáticas humanas en células madre pluripotentes inducidas (iPSCs) utilizando el vector retroviral con las buenas prácticas agrarias

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparación de las células embrionarias de fibroblastos de ratón adecuados para el cultivo de células estaminales embrionarias humanas pluripotentes inducidas, y

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Generación de las células madre pluripotentes inducidas a partir de sangre periférica mediante el STEMCCA vector lentiviral

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

La generación de cardiomiocitos humanos: Protocolo de diferenciación de células madre pluripotentes inducidas humanas Feeder libres

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente generación, por actividad humana células madre pluripotentes de células somáticas humanas con virus Sendai-

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.