1. Reprogramación <ol><li> Las células de las semillas BJ a una densidad de 1 x 10 5 células por pocillo de una placa de 6 pocillos. Cultivar las células en 2 ml de medio de cultivo (450 ml EMEM suplementado con 50 ml de FBS ES cualificado, 5 ml de 10 mM no aminoácidos esenciales, 5 ml de penicilina / estreptomicina, 5 ml de 200 mM L-glutamina y 0,9 ml de 55 mM β -mercaptoetanol) durante la noche a 37 ° C y 5% de CO 2. </li><li> El mismo día, empezar a preparar platos MEF alimentador mediante la adición de 0,1% de gelatina diluida en agua a una placa de 6 pocillos e incubar durante la noche a 37 ° C y CO 2 al 5%. </li><li> Después de la incubación, retire medio de las células BJ y añadir 2 ml de medio de cultivo suplementado con 6 mg / ml de polibreno, 500 l hOct4-lentivirus, 50 l hSox2-lentivirus, 50 l hNanog lentivirus y 50 l hLin28 lentivirus-a cada pocillo . Agite suavemente la placa para asegurar una capa uniforme del medio. Incubar toda la noche a 37 ° C y CO 2 al 5%. </li><li> El mismo día, eliminar un vial de CF-1 en las células MEF a partir de nitrógeno líquido y descongelación. Retire el líquido de los pozos recubiertos de gelatina (ver paso 1.2) y añadir las células MEF alimentador a una densidad de 0,2 x 10 5 células / pocillo. El volumen total por pozo debe ser 2 ml de células en un medio de crecimiento MEF (450 ml DMEM suplementado con FBS 50 ml y 5 ml no aminoácidos esenciales). </li><li> Al día siguiente, separar las células BJ con 0,05% de tripsina / EDTA y se centrifuga a 200 xg durante 5 minutos. Aspirar el medio y resuspender en medio de crecimiento. Quitar medio de la placa de alimentación MEF (ver paso 1.4) y añadir 2 ml / pocillo de la suspensión de células BJ. La concentración de células debe ser de aproximadamente 5 x 10 4 células /. Incubar toda la noche a 37 ° C y CO 2 al 5%. </li><li> Vuelva a colocar medio 24 horas después de la resiembra con humanos ES / iPS medio de cultivo celular (400 ml DMEM/F12 complementado con 100 ml de suero de reemplazo golpe de gracia, 5 ml de 10 mM no aminoácidos esenciales, 5 ml de 200 mM L-glutamina, 0,9 ml 55 mM β-mercaptoetanol y 20 ng / ml de bFGF recombinante humana). Cambio de medio cada 24 horas durante 7 días. </li><li> Después de siete días, el cambio medio a medio MEF acondicionado (ver sección 2). </li></ol> 2. Preparación del medio de MEF acondicionado <ol><li> Semilla CF-1 en células alimentadoras MEF 2 x 10 5 células / pocillo en una placa de 6 y medio de crecimiento en 2 ml MEF. Incubar toda la noche a 37 ° C y CO 2 al 5%. </li><li> Cambio de medio para la salud humana ES / iPS medio de cultivo celular. Incubar toda la noche a 37 ° C y CO 2 al 5%. </li><li> Recoger el líquido sobrenadante cada 24 horas durante cuatro días. Filtrar el sobrenadante a través de un filtro de 0,22 micras. </li><li> Suplemento sobrenadante con 50 ng / ml de bFGF. </li></ol> 3. iPS selección de colonias y pases <ol><li> Seleccione una colonia y como ES-re de semillas en el medio de la cultura humana ES / IPS suplementado con 10 mM de Stemolecule Y27632 en placas de cultivo de células pre-siembra con CF-1 en las células MEF alimentador. Incubar toda la noche a 37 ° C y CO 2 al 5%. </li><li> Cambiar el medio de cultivo cada 24 horas durante los primeros siete días (sin complementar con Stemolecule Y27632). Después de siete días, el cambio medio a medio MEF acondicionado (véase la sección 2 de la preparación). Continuamos pases de las células hasta que presentaron la morfología típica madre embrionarias humanas. </li></ol> 4. El examen de inmuno marcadores de pluripotencia <ol><li> Lave suavemente las células tres veces con PBS. </li><li> Fijar las células con 500 l de solución fijadora durante 20 minutos a temperatura ambiente. </li><li> Lave suavemente las células tres veces con PBS. </li><li> Bloquear la unión no específica con 500 l de amortiguación de bloqueo durante una hora a temperatura ambiente. </li><li> Se incuban las células con 250 ml del anticuerpo primario específico la noche a 4 ° C (en el ejemplo TRA-1-81, TRA-1-60, SSEA-4, SSEA-3, Oct4, Sox2, y Nanog Lin28 a 1:100 diluciones). </li><li> Lave suavemente las células tres veces con PBS. </li><li> Se incuban las células con 250 l de anticuerpo secundario durante 1 hora a temperatura ambiente, manteniéndose alejado de la luz (en el ejemplo de cabra anti-ratón conjugado IgM Cy3, cabra anti-ratón IgG Cy3 conjugado de cabra anti-rata Cy3 IgM conjugado y de cabra anti- conejo Cy3 conjugado a 1:300 diluciones). </li><li> Lave suavemente las células tres veces con PBS. </li><li> Añadir DAPI (concentración final de 1 mg / ml) para el último lavado e incubar 5 minutos para visualizar los núcleos. </li><li> Analizar las células con una lupa. </li></ol> Parte 5: Resultados de Representante 1. Resultados Morfología: Fibroblastos de prepucio humano (BJ), las células fueron co-transducidas con Oct4, Sox2, Nanog, y Lin28. Los cambios morfológicos que se observaron a partir del día 4 post-transducción, y el grupo de células se volvieron más apretada en el día 17 (Figura 1). Colonias fueron recogidos manualmente en el día 25 y se cultivaron en CF-1 células alimentadoras MEF. Para facilitar la formación de colonias de células iPS tras la reprogramación, se utilizó Stemolecule Y27632, Un inhibidor de ROCK, para la siembra inicial de la noche en cada paso. colonias de células iPS con buena morfología se observaron después de tres rondas secuenciales de la colonia de la cosecha y pasajes. 2. Expresión de marcadores de pluripotencia: Para caracterizar mejor las colonias de células iPS aislados, se buscó la presencia de marcadores de pluripotencia común se expresa en células madre embrionarias. Las colonias mostraron una fuerte fosfatasa alcalina (AP) actividad (Figura 2). Además, inmunocitoquímica (CPI) se realizó en las colonias de células iPS con un panel de anticuerpos marcadores específicos de pluripotencia, incluidos los marcadores de la superficie de TRA-1-81, TRA-1-60, SSEA-4 y SSEA 3, así como marcadores nucleares Oct4, Sox2 y Nanog. El colonias aisladas iPS fueron positivos para todos los marcadores (Figura 3). Los resultados de la CPI muestran que las células iPS mostró el patrón adecuado pluripotencia expresión de los marcadores, lo que demuestra que estas células iPS se parecen mucho a indiferenciado de células madre embrionarias humanas. <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="Figura 1" /> Figura 1: Los cambios morfológicos de las células transducidas BJ. Imágenes brillantes de campo de una colonia de células iPS típico formado en (A) 4 días y (B) 17 días después de la transducción. <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="Figura 2" /> Figura 2: La actividad de AP de reprogramar las células BJ. Tres diferentes colonias teñidas con Stemgent Kit de tinción fosfatasa alcalina. <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="Figura 3" /> Figura 3: Las células iPS expresan altos niveles de los marcadores de células ES siguientes específicos: los marcadores de superficie TRA-1-81, TRA-1-60, SSEA-4 y SSEA-3, y marcadores nucleares de 04 de octubre, Nanog y Sox2.

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thompson, 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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (1917).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Koyanagi, M., Takahashi, J., Arakawa, Y., Doi, D., Fukuda, H., Hayashi, H., Narumiya, S., Hashimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+Rho/ROCK+pathway+reduces+apoptosis+during+transplantation+of+embryonic+stem+cell-derived+neural+precursors.">Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.</a> J. Neurosci. Res. 86, 270-280 (2008).</li></ol>

The authors of this article are employed by Stemgent that produces reagents and instruments used in this article.

Estos resultados demuestran que la Stemgent Set Humanos Lentivirus TF puede ser utilizado para generar de manera eficiente las colonias de iPS mediante la inducción de la expresión ectópica de transducidas factores de transcripción en fibroblastos humanos. En el diseño de experimentos de reprogramación, las variables se deben considerar varias para optimizar la eficiencia de la reprogramación. En primer lugar, la participación activa de virus al objetivo ratio (multiplicidad de infección MOI) puede ser necesario modificar durante el paso de la transducción de primaria para lograr la eficiencia de transducción óptima. En segundo lugar, las condiciones de crecimiento de las células diana pueden afectar la reprogramación. Las células sanas y proliferantes son más susceptibles a la reprogramación. En tercer lugar, al modificar el protocolo para el número de células diferentes, se recomienda que los números de las células diana se ajustará proporcionalmente a la superficie de la placa de cultivo. Por último, la aplicación de inhibidores de rock como Y27632 deben ser considerados para ayudar a asegurar la reprogramación exitosa ya que estudios recientes han demostrado su utilidad en la mejora de la supervivencia de la colonia hES 4,5.

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		<th>Catalogue Number</th>
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<td>Human TF Lentivirus Set</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>00-0005</td>
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<td>Stemolecule Y27632</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>04-0012</td>
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<td>TRA-1-81 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0012</td>
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<td>TRA-1-60 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
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<td>SSEA-4 antibody</td>
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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

En 2006, Yamanaka y sus colegas demostraron por primera vez que retrovirus mediada por la entrega y la expresión de Oct4, Sox2, c-Myc y Klf4 es capaz de inducir el estado pluripotente en fibroblastos de ratón. 1 El mismo grupo también informó de la reprogramación exitosa de las células somáticas humanas en madre pluripotentes inducidas (iPS) utilizando células versiones humanas de los mismos factores de transcripción entregada por vectores retrovirales. 2 Por otra parte, James Thomson et al. informaron que el lentivirus mediada por la co-expresión de otra serie de factores (Oct4, Sox2, y Nanog Lin28 ) fue capaz de reprogramar las células somáticas humanas en células iPS. 3 Las células iPS son similares a las células madre embrionarias en la morfología, la proliferación y la capacidad de diferenciarse en todos los tipos de tejidos del cuerpo. Células iPS humanas tienen una clara ventaja sobre células madre embrionarias, ya que muestran las características clave de células madre embrionarias sin el dilema ético de la destrucción de embriones. La generación de pacientes específicos de las células iPS evita un obstáculo importante para las terapias personalizadas medicina regenerativa mediante la eliminación de la posibilidad de rechazo inmunológico no autólogo las células trasplantadas. Aquí se demuestra el protocolo para la reprogramación de células de fibroblastos humanos con el Stemgent Set Humanos Lentivirus TF. También muestran que las células reprogramadas con este conjunto comienza a mostrar iPS morfología cuatro días después de transducción. Utilizando el Stemolecule Y27632, que hemos seleccionado para las células iPS y la morfología correcta observado después de tres rondas secuenciales de la colonia de la cosecha y pasajes. También demuestran que después de reprogramar las células muestran la pluripotencia marcador de AP, los marcadores de superficie TRA-1-81, TRA-1-60, SSEA-4, y SSEA-3, y marcadores nucleares Oct4, Sox2 y Nanog.

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set at JoVE.com

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

Demostramos el protocolo para la generación de células madre pluripotentes inducidas a partir de células somáticas humanas con lentivirus mediada por la entrega de los factores humanos Oct4, Sox2, Nanog, y Lin28. Pluripotencia fue confirmada por la morfología y la presencia de células madre embrionarias (ES) de células específicas de los marcadores.

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 ...

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Unit 1

Embryonic and Induced Pluripotent Stem Cells

SCIENTISTS IN ACTION

29.7K vistas.  Stemgent. Demostramos el protocolo para la generación de células madre pluripotentes inducidas a partir de células somáticas humanas con lentivirus mediada por la entrega de los factores humanos Oct4, Sox2, Nanog, y Lin28. Pluripotencia fue confirmada por la morfología y la presencia de células madre embrionarias (ES) de células específicas de los marcadores.

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

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

La cultura y el mantenimiento de células estaminales embrionarias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Investigación

Biología

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Generación de células madre pluripotentes inducidas por reprogramación de fibroblastos embrionarios de ratón con un factor de transcripción Cuatro, doxiciclina inducible sistema de transducción de lentivirales

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

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 ...

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 ...

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 ...

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 ...