Estos estudios fueron financiados en parte por el NIH UO1HL107443-01 Premio a GJM y GM.

1. El aislamiento y la expansión de las células mononucleares de sangre periférica (PBMCs) DÍA 0 <ol><li> Dibujar 4 ml de sangre periférica en un tubo BD Vacutainer CPT Preparación célula con citrato de sodio. Invertir el tubo de 8 a 10 veces y centrifugar a 1.800 xg durante 30 min a temperatura ambiente. Idealmente, este paso se debe hacer dentro de 2 horas de recogida. </li><li> Recoger las células mononucleares (MC) pipeteando la capa leucocitaria (capa de células de barrera entre el gel y plasma) en un tubo estéril de 15 ml de centrífuga cónico. Llevar a un volumen total de 10 ml estéril con salina tamponada con fosfato (PBS), invertir varias veces y centrifugar a 300 xg durante 15 min. </li><li> Resuspender las células en 10 ml de PBS estéril y realizar el recuento de células. Transferir 1 a 2x10 6 células en un tubo de centrífuga estéril de 15 ml cónico y se centrifuga a 300 xg durante 10 min. </li><li> Resuspender las células en 2 ml de medio de expansión (EM) (QBSF-60 Stem medio celular que contenía 50 mg / ml AscoÁcido RBIC, 50 ng / ml de SCF, 10 ng / ml de IL-3, 2 U / ml de EPO, 40 ng / ml de IGF-1, dexametasona 1 mM y 100 mg / ml o primocin 1% Pen / Strep) y transferir a un pocillo de una placa de 12 pocillos. Se incuban las células en un 37 ° C, 5% de CO 2 incubadora. </li><li> Centrifugar las células restantes a 300 x g durante 10 min y congelación ~ 2x10 6 células / vial en FBS que contenía 10% de DMSO. </li><li> Para iniciar el protocolo utilizando PBMCs congeladas, descongelar 1 vial de células en 10 ml de medio de QBSF y centrifugar a 300 xg durante 10 min. Resuspender las células en 2 ml de EM y transferir a un pocillo de una placa de 12 pocillos. Se incuban las células en un 37 ° C, 5% de CO 2 incubadora. </li></ol> Los días 3 y 6 <ol start="17"><li> Transferir las células a un tubo estéril de 15 ml cónico y lavar el bien una vez con 1 ml de QBSF-60 Media de células madre para recoger las células adherentes. </li><li> Centrifugar las células a 300 xg durante 10 min. </li><li> Resuspender las células en 2 ml de EM y transferir a un pocillo de un 12Y placa. Se incuban las células en un 37 ° C, 5% de CO 2 incubadora. </li></ol> 2. La transducción de las PBMCs con STEMCCA Lentivirus DÍA 9 <ol><li> Transferir las células a un tubo estéril de 15 ml cónico y lavar el bien una vez con 1 ml de QBSF-60 Media de células madre para recoger las células adherentes. </li><li> Centrifugar las células a 300 xg durante 10 min. </li><li> Resuspender las células en 1 ml de EM fresco que contenía 5 mg / ml de polibreno y lentivirus STEMCCA (MOI = 1 a 10) y la transferencia a un pocillo de una placa de 12 pocillos. (El protocolo que describe la producción de partículas lentivirales se puede encontrar en <a href="http://www.bumc.bu.edu/stemcells/protocols/" target="_blank">http://www.bumc.bu.edu/stemcells/protocols/</a> ) </li><li> Se centrifuga la placa a 2.250 rpm a 25 ° C durante 90 min. </li><li> Después de girar, agregar un adicional de 1 ml de EM frescas que contiene 5 g / ml de polibreno durante un total de 2 ml de medio y se incuba la placa en un 37 ° C,5% de CO 2 incubadora. </li></ol> DÍA 10 <ol start="16"><li> Transferir las células a un tubo estéril de 15 ml cónico y lavar el bien una vez con 1 ml de QBSF-60 Media de células madre para recoger las células adherentes. </li><li> Centrifugar las células a 300 xg durante 10 min. </li><li> Resuspender las células en 2 ml de EM y transferir a 1 pocillo de una placa de 12 pocillos. Se incuban las células en un 37 ° C, 5% de CO 2 incubadora. </li></ol> 3. Revestimiento células transducidas en MEFs DÍA 11 <ol><li> Recubrir los pocillos de una placa de 6 pocillos con 0,1% de gelatina y fibroblastos placa inactivadas embriones de ratón (MEFs) a 2x10 5 células / pocillo en medio de MEF (IMDM que contenía 10% de FBS, 1% no aminoácidos esenciales, 100 mM β- mercaptoetanol, 2 mM L-glutamina, 100 mg / ml o primocin 1% Pen / Strep). Prepare 3 pozos por infección. </li></ol> DÍA 12 <ol start="2"><li> Transferir las células a un tubo estéril de 15 ml cónico<html> y lavar el pozo una vez con 1 ml de QBSF-60 Media de células madre para recoger las células adherentes. </li><li> Resuspender las células en 3 ml de medio de MEF que contiene 10 ng / ml de bFGF, y ácido ascórbico y los factores de crecimiento en las mismas concentraciones que se utilizan en EM media (50 mg / ml de ácido ascórbico, 50 ng / ml de SCF, 10 ng / ml IL -3, 2 U / ml de EPO, 40 ng / ml de IGF-1, 1 mM de dexametasona). </li><li> Placa de 1 ml de células por pocillo de una placa de 6 pocillos que contenía MEFs. Añadir 1,5 ml de MEF medios de comunicación con el bFGF, ácido ascórbico, y factores de crecimiento para un total de 2,5 ml de medio / pocillo. </li><li> Se centrifuga la placa a 500 rpm a 25 ° C durante 30 min. Se incuba la placa en un 37 ° C, 5% de CO 2 incubadora. </li></ol> DÍA 14 <ol start="6"><li> Células de alimentación cada dos días con 2,5 ml de medio de MEF que contienen 10 ng / ml de bFGF y 50 ug / ml de ácido ascórbico (sin factores de crecimiento). Aspirar y desechar las células flotantes con cada toma. </li><li> Añadir MEFs adicional cuando sea necesario (generalmente una vez a la semana). </li></ol>p &quot;&gt; ~ DÍA 20 <ol start="8"><li> Una vez que aparecen pequeñas colonias, las células de alimentación diaria con 2 ml de células madre embrionarias humanas (hESC) medio (DMEM/F12 que contiene reemplazo del 20% Knockout Serum, 1% no aminoácidos esenciales, 100 mM β-mercaptoetanol, 2 mM L-glutamina , 100 mg / ml o primocin 1% Pen / Strep y 10 ng / ml de bFGF). </li></ol> 4. Picking y expansión de clones de IPSC ~ DÍA 30 a 40 <ol><li> Recoger cada colonia en pocillos individuales de una placa de 12-así pre-siembra con MEFs inactivada que contiene 1 ml / pocillo de medio de hESC más 10 mM de Stemolecule Y27632 (ROCK inhibidor). </li><li> Alimentar a las células al día posteriormente con 1 ml de medio de hESC sólo (sin inhibidor ROCK). </li></ol> 5. La tinción de inmunofluorescencia para marcadores de pluripotencia <ol><li> La tinción se realizó utilizando el kit &quot;célula ES Marker Kit de muestra&quot; de Millipore y siguiendo el protocolo del fabricante. </li></ol> 6. Escisión de Integnominal STEMCCA Vector <ol><li> Escisión de reprogramación casete se logra utilizando un Cre-IRES-Puro expresando vector de transfección y tras breve selección puromicina como se describe 6. </li></ol>

<ol>
	<li>Lowry, W. E., Richter, L., Yachechko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Park, I. H., Zhao, R., West, J. 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=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Takahashi, K., Tanabe, K., Ohnuki, 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=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., 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=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 (2007).</li><li>Stadtfeld, M., Hochedlinger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotency:+history,+mechanisms,+and+applications.">Induced pluripotency: history, mechanisms, and applications.</a> Genes Dev. 24, 2239-2263 (2010).</li><li>Somers, A., Jean, J. C., Sommer, C. 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=Generation+of+transgene-free+lung+disease-specific+human+induced+pluripotent+stem+cells+using+a+single+excisable+lentiviral+stem+cell+cassette.">Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.</a> Stem Cells. 28, 1728-1740 (2010).</li><li>Loh, Y. H., Hartung, O., Li, 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=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., Yuasa, S., Oda, 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=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., Dawlaty, M. M., Gao, Q., 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=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Chou, B. K., Mali, P., Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+human+iPS+cell+derivation+by+a+non-integrating+plasmid+from+blood+cells+with+unique+epigenetic+and+gene+expression+signatures.">Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.</a> Cell Res. 21, 518-529 (2011).</li><li>vanden Akker, E., Satchwell, T. J., Pellegrin, S., Daniels, G., Toye, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+the+in+vitro+erythroid+expansion+potential+resides+in+CD34(-)+cells,+outweighing+the+contribution+of+CD34(+)+cells+and+significantly+increasing+the+erythroblast+yield+from+peripheral+blood+samples.">The majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.</a> Haematologica. 95, 1594-1598 (2010).</li></ol>

No hay conflictos de interés declarado.

Nos en la presente memoria se describe el uso del vector lentiviral STEMCCA para generar iPSCs humanos a partir de células mononucleares aisladas de unos pocos mililitros de sangre periférica recién recogida. El protocolo también se puede utilizar para reprogramar PBMCs congeladas (obtenidos directamente a partir de la capa leucocitaria), un detalle de importantes implicaciones prácticas cuando se utilizan células de donantes adquiridos desde una ubicación distante. Antes de la inducción de la reprogramación, P...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> BD Vacutainer CPT Celular Preparación del tubo con citrato de sodio </td><td> BD Biosciences </td><td> 362760 </td><td></td></tr><tr><td> QBSF-60 Stem medio celular </td><td> De calidad biológica </td><td> 160-204-101 </td><td></td></tr><tr><td> IMDM </td><td> Invitrogen </td><td> 12440 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td><td></td></tr><tr><td> FBS </td><td> Atlanta Biológicos </td><td> S10250 </td><td></td></tr><tr><td> Knockout Serum reemplazo </td><td> Invitrogen </td><td> 10828 </td><td></td></tr><tr><td> Primocin </td><td> Invivogen </td><td> ant-pm-2 </td><td></td></tr><tr><td> Pen / Strep </td><td> Invitrogen </td><td> 15140 </td><td></td></tr><tr><td> L-Glutamina </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> No esenciales Aminoácidos </td><td> Invitrogen </td><td> 11140 </td><td></td></tr><tr><td> β-mercaptoetanol </td><td> MP Biomedicals </td><td> 190242 </td><td></td></tr><tr><td> Ácido ascórbico </td><td> Sigma </td><td> A4544 </td><td></td></tr><tr><td> IGF-1 </td><td> R &amp; D Systems </td><td> 291-G1 </td><td></td></tr><tr><td> IL-3 </td><td> R &amp; D Systems </td><td> 203-IL </td><td></td></tr><tr><td> SCF </td><td> R &amp; D Systems </td><td> 255-SC </td><td></td></tr><tr><td> EPO </td><td> R &amp; D Systems </td><td> 286-EP </td><td></td></tr><tr><td> Dexametasona </td><td> Sigma </td><td> D4902 </td><td></td></tr><tr><td> Polybrene </td><td> Sigma </td><td> H-9268 </td><td></td></tr><tr><td> bFGF </td><td> R &amp; D Systems </td><td> 233-FB </td><td></td></tr><tr><td> Stemolecule Y27632 </td><td> Stemgent </td><td> 04-0012 </td><td></td></tr><tr><td> ES Cell Marker Kit de muestra </td><td> Millipore </td><td> SCR002 </td><td></td></tr></tbody></table>

generation of human induced pluripotent stem cells from peripheral blood using the stemcca lentiviral vector

A través de la expresión ectópica de cuatro factores de transcripción, Oct4, Klf4, Sox2 y cMyc, las células somáticas humanas se puede convertir en un estado pluripotente, generando así llamadas células madre pluripotentes inducidas (iPS) 1-4. Específicos del paciente iPSCs carecen de las preocupaciones éticas que rodean a las células madre embrionarias (CME) y pasaría por alto el rechazo inmunológico posible. Por lo tanto, iPSCs han atraído una considerable atención por los estudios de modelos de enfermedades, la detección de compuestos farmacológicos y terapias regenerativas 5. Hemos demostrado la generación de transgén libres de iPSCs humanos de pacientes con enfermedades pulmonares diferentes usando una sola extirpable policistrónico Stem lentiviral casete celular (STEMCCA) que codifica los factores de Yamanaka 6. Estas líneas IPSC se generaron a partir de fibroblastos de la piel, el tipo de célula más común utilizado para la reprogramación. Normalmente, la obtención de fibroblastos de piel requiere un punzón de biopsia seguida por la expansión de la células en la cultura de algunos pasajes. Es importante destacar que, un número de grupos han informado de la reprogramación de células humanas de sangre periférica en iPSCs 7-9. En un estudio, una versión inducible Tet del vector STEMCCA se empleó 9, que requiere las células de la sangre que se infectan simultáneamente con un lentivirus constitutivamente activa que codifica el transactivador inverso tetraciclina. En contraste con los fibroblastos, las células de sangre periférica pueden recogerse a través de procedimientos mínimamente invasivos, reduciendo considerablemente el malestar e incomodidad del paciente. Un protocolo sencillo y eficaz para la reprogramación de células de la sangre utilizando un vector constitutiva sujetos a impuestos especiales solo pueden acelerar la aplicación de la tecnología IPSC para hacerla accesible a una comunidad de investigación más amplia. Además, la reprogramación de células de sangre periférica permite la generación de iPSCs de individuos en los que las biopsias de piel debe ser evitado (es decir. Aberrante cicatrización) o debido a condiciones de enfermedad pre-existentes Preventing acceso a biopsias. Aquí se demuestra un protocolo para la generación de iPSCs humanos a partir de células mononucleares de sangre periférica (PBMCs) utilizando una única floxed-extirpable vector lentiviral que expresa constitutivamente los 4 factores. PBMC recién arrancadas o descongelados se expanden durante 9 días descrita 10,11 en ácido ascórbico que contiene medio, SCF, IGF-1, IL-3 y EPO antes de ser transducidas con el lentivirus STEMCCA. Después las células se sembraron en MEFs y ESC-como colonias se puede visualizar dos semanas después de la infección. Finalmente, los clones seleccionados se expanden y se ensayaron para la expresión de los marcadores de pluripotencia SSEA-4, Tra-1-60 y Tra-1-81. Este protocolo es simple, robusto y muy consistente, proporcionando una metodología fiable para la generación de iPSCs humanos fácilmente accesible desde 4 ml de sangre.

Se demuestra un protocolo simple y eficaz para la generación de iPSCs humanos a partir de PBMCs utilizando un vector lentiviral único. Figura 1A muestra una representación esquemática del protocolo. La sangre se recogió en un tubo BD Vacutainer CPT Preparación célula con citrato de sodio, y después de la centrifugación, las células mononucleares se pueden recoger de la interfaz entre el gel de poliéster y el plasma (buffy coat) (Figura 1B). Las PBMC aisladas se expanden en cu...

Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

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

Aquí se muestra un protocolo simple y eficaz para la generación de iPSCs humanos de 3-4 ml de sangre periférica utilizando un único vector lentiviral reprogramación. La reprogramación de células de la sangre fácilmente disponibles promete acelerar la utilización de la tecnología de IPSC para hacerla accesible a una comunidad de investigación más amplia.

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, ...

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Biology

26.4K vistas.  Boston University School of Medicine. Aquí se muestra un protocolo simple y eficaz para la generación de iPSCs humanos de 3-4 ml de sangre periférica utilizando un único vector lentiviral reprogramación. La reprogramación de células de la sangre fácilmente disponibles promete acelerar la utilización de la tecnología de IPSC para hacerla accesible a una comunidad de investigación más amplia.

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

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 ...

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 ...

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 ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Generación rápida y eficiente de las neuronas a partir de células madre pluripotentes en un formato de placa Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.