Este trabajo fue apoyado por la Ataxia de Friedreich Research Alliance y una donación de la familia de Arnold piloto de la Fundación y el Centro de Células Madre y Biología del Desarrollo en el MD Anderson Cancer Center.

1. Virus de la producción y la transducción <ol><li> Placas Phoenix anfo células a una densidad de ~ 7-8x10 6 por placa de 10 cm en 10 ml de medio DMEM (glucosa DMEM alto, 10% de FBS inactivado calor, 2 mM de L-glutamina, sin antibióticos). Lugar en la incubadora y la cultura durante toda la noche a 37 ° C, 5% de CO 2. </li><li> Al día siguiente, transfección de células de Phoenix con 12 mg de un vector de codificación sea Oct3 / 4, Sox2, Klf4, c-myc, o gen GFP (Addgene plásmidos 17217, 17218, 17219, 17220) y 35 l Fugene 6. Prepare la mezcla de transfección en 500 l de medio DMEM (glucosa DMEM alto, 2 mM L-glutamina, sin SFB y antibióticos). Incubar 20 minutos. Pipetear con cuidado los complejos de ADN en las placas de 10 cm que contienen 70-80% confluentes células de Phoenix. </li><li> Vuelva a colocar los medios de comunicación 6 a 8 horas después de la transfección con medio DMEM (glucosa alta DMEM, 10% al calor de SFB inactivado, 2 mM L-glutamina), que contiene la penicilina y la estreptomicina. </li><li> Posteriormente, recoger el virus que contiene los medios 4 veces en 12 hintervalos, se combinan todas las partes y se filtra utilizando un filtro de 0,45 micras para eliminar las células desprendidas y escombros (los medios de comunicación que contienen partículas virales se pueden mantener en el refrigerador por 2 semanas sin perder la actividad infecciosa, sin embargo, no se recomienda la congelación). </li><li> Para retrovirales transducción, los fibroblastos humanos adultos de la placa (paso 10, Coriell Laboratories) de un stock congelado 24 horas anteriores al último día de recolección de los medios de comunicación retroviral. Semillas de las celdas de 6-así gelatina placas cubiertas a la densidad 1x10 5 células por pocillo en DMEM glucosa alta, 10% de FBS, 2 mM de L-glutamina, penicilina, estreptomicina y no esenciales aminoácidos. </li><li> El sustituto día siguiente medio DMEM con medios que contienen partículas virales obtenidos a partir de células de Phoenix. Añadir los medios de comunicación viral (1 ml de cada Oct4, Sox2, Klf4 y c-myc los medios de comunicación, 4 ml en total) suplementado con 6 mg / ml de polibreno en cada pocillo de la placa de 6 pocillos de los cultivos de fibroblastos y centrifugar a 1600 g de 1 hora a 20 ° C. Aproximadamente 12 horas after de la transducción, sustituir a los medios de comunicación virales con medio de fibroblastos cultura. Realizar la infección viral y centrifugación 3 veces en intervalos de 24 horas. </li><li> Fibroblastos de cultivo en medio DMEM durante 48 horas después de la última infección. Controlar la eficacia de la infección por transducciones paralelas con un medio de retrovirales que expresan GFP. La Figura 1 ilustra la eficacia de centrifugación-facilitado infección retroviral de fibroblastos humanos. </li></ol> 2. Reprogramación <ol><li> Preparar placas de 6 pocillos con γ-irradiados capas alimentadoras MEF sembrando células MEF a una densidad de ~ 2x10 5 células por pocillo de la placa de gelatina tratada 6 pocillos, en medio de crecimiento de fibroblastos. El día siguiente dividir fibroblastos humanos infectados con 0,05% de tripsina / EDTA y semillas ellos en la densidad de 1.2x10 ~ 4 por pocillo en el medio de crecimiento de fibroblastos. </li><li> Al día siguiente, reemplazar a los medios de comunicación con HES medios de comunicación (DMEM/F12, la sustitución del 20% de suero golpe de gracia, no aminoácidos esenciales, penicillin / estreptomicina, 2 mM L-glutamina, 0,1 mM β-mercaptoetanol, 20 ng / ml de factor de crecimiento de fibroblastos basal (bFGF), suplementado con 0,5 mM butirato sódico durante los primeros 7 días de la reprogramación. Cambiar los medios de comunicación todos los días. </li><li> Monitorear los cambios morfológicos en las células transducidas todos los días. Las colonias de las caderas-como las células comenzarán a surgir aproximadamente 5 - 10 días después de la transferencia de los fibroblastos en células transducidas de alimentación. Las colonias hiPSC están listos para ser aislado aproximadamente 14 - 28 días después de placas ellos en las células alimentadoras MEF. </li></ol> 3. El aislamiento de colonias hiPSC <ol><li> Un día antes de recoger las colonias hiPSC preparar placas de 24 pocillos con la γ-células irradiadas MEF alimentador (aproximadamente 4x10 4 células por pocillo) en medio de crecimiento de fibroblastos. En esta etapa colonias hiPSC también pueden ser transferidos a la cultura alimentador gratis con Matrigel y medio mTeSR1. </li><li> Antes de aislamiento de colonias hiPSC preparar placas MEF mediante la eliminación de los fibroblastos crecimiento medium y aclarado MEFs con PBS para eliminar las trazas de FBS. Posteriormente añadir 0,5 ml hES medio (o mTeSR1 medio en el caso de Matrigel pocillos recubiertos) con 10 mM inhibidor ROCA Y27632 a cada pocillo 7,8. </li><li> Si es necesario, eliminar los fibroblastos que rodean las colonias hiPSC con una aguja de calibre 21 bajo un microscopio montado en una campana de flujo laminar (Figura 2A - C). Lavar las placas con PBS y añadir frescos medios hES que contienen el anticuerpo específico Tra-1-81 StainAlive (1:200, Stemgent). Después de 30 min., Reemplazar medios que contienen el anticuerpo con frescos medios hES suplementado con 10 mM inhibidor ROCA. Examinar las placas bajo el microscopio de fluorescencia y la marca de TRA-1-81 colonias positivas utilizando un marcador objetivo (Figura 2D). </li><li> Cortar los Tra-1-81 colonias positivas hiPSC bajo un microscopio en una campana laminar, en varias piezas pequeñas con una aguja de calibre 21 como se muestra en la Figura 2E y F. </li><li> Usando una pipeta automática (P200) de transferencia de fragmentos de colonias en hiPSC individual pozos de la placa de 24 pocillos con MEFs o Matrigel. Evitar la transferencia de las células que no son las caderas. Coloque las placas en un 37 ° C, 5% de CO 2 incubadora y permitir que las colonias hiPSC para conectar de 24-36 h. </li><li> Cambio hES o mTeSR1 (para las colonias cultivadas en Matrigel) los medios de comunicación todos los días. Las colonias con hiPSC correcta HES-como la morfología será visible 48 horas después de la transferencia inicial sobre una placa de 24 pocillos. </li><li> Manual de paso hiPSC colonias cada 6 - 8 días sobre un 12-así y, posteriormente, en una placa de 6 pocillos. </li><li> Después de la expansión clonal y establecer las líneas hiPSC, analizar la expresión de marcadores de pluripotencia TRA-1-60, SSEA-4, Oct3 / 4, Sox2 y Nanog utilizando inmunocitoquímica como se muestra en la Figura 3. Evaluar la integridad genómica y potencial de diferenciación de las líneas obtenidas mediante cariotipo y análisis de la formación de teratomas 6,9. </li></ol> 4. Los resultados representativos Transducción eficiente con retrovirus que contiene los medios de comunicación is crítico para el éxito reprogramación. Se recomienda llevar a cabo toda la transfección / infección procedimiento utilizando un GFP expresando virus cada experimento individual reprogramación para supervisar la eficiencia, como se muestra en la Figura 1. El título del virus GFP expresar determinado, como se describe en 10, a través de la transducción de fibroblastos humanos utilizando no concentrados medios virales era típicamente en el intervalo de 0,5 a 5 x 10 7 partículas virales por ml (VP / ml). Los fibroblastos cambiar la morfología como a los 2 días después de la última infección. Trypsinized fibroblastos humanos deben ser cuidadosamente contarán antes de la siembra de ellos en las células alimentadoras del MEF. Se recomienda a las células de semillas a 3 densidades diferentes (6x10 3, 4 1.2x10, 2.5x10 4 por un solo pocillo de una placa de 6 pocillos), ya que cada línea celular se muestra característica de crecimiento diferente y densidad de siembra es crítico para la eficiencia de reprogramación. Butirato sódico utilizado para elinicial 7 - 14 días de reprogramación aumenta la eficiencia de la formación de hiPSC aproximadamente 5 veces. Con frecuencia, especialmente cuando los fibroblastos infectados se sembraron a una densidad más alta, las células similares a fibroblastos pueden recubrir una placa de cultivo y cubrir colonias hiPSC como se muestra en la Figura 2A. En este caso, la capa de fibroblastos puede ser cuidadosamente levantado y retirado para descubrir colonias hiPSC (Figura 2B). Posteriormente, las colonias hiPSC debe enjuagarse con HES medios y se tiñeron con Tra-1-81 anticuerpo. Dependiendo de las células de fibroblasto, aproximadamente 20 a 40% de las colonias que demuestran con CMPi-como morfología no se tiñen con Tra-1-81 anticuerpo. Identificadas colonias hiPSC puede ser transferido de una placa dentro de la próxima 12 - 24h. Incubación prolongada dará lugar a la diferenciación rápida de hiPSCs. Las colonias pueden ser manualmente pasajes en cualquiera de las células alimentadoras MEF o placas Matrigel recubiertos. Después de la expansión establecida clones hiPSC deben ser evaluados con inmunocitoquímica (CPI) para la expresión de pluripotenCY marcadores como se demuestra en la Figura 3. Además, la caracterización molecular detallada de las líneas de células iPS generadas debe incluir: análisis de la expresión génica pluripotencia mediante RT-PCR, la demostración de la desmetilación del ADN a los promotores de los genes de pluripotencia y análisis de los transgenes de silenciamiento 9. <img alt="Figura 1" src="/files/ftp_upload/3416/3416fig1.jpg" /> Figura 1. La eficiencia de la transducción viral determinó a través de los retrovirus que expresa GFP. (A, B) fibroblastos humanos adultas derivadas de los pacientes la ataxia de Friedreich (GM03665, Coriell repositorio) se visualizaron después de dos consecutivos con los medios de comunicación las infecciones retrovirales las buenas prácticas agrarias. (C, D) fibroblastos humanos fueron infectados con el mismo lote de los medios de GFP retrovirales seguido por centrifugación de las células directamente sobre las placas 6-y durante 1 hora a 1600g. Las imágenes fueron capturados 48h después de la infección. <img alt= &quot;Figura 2&quot; src = &quot;/ files/ftp_upload/3416/3416fig2.jpg&quot; /&gt; Figura 2. Identificación y aislamiento de colonias hiPSC. (A) de la imagen de contraste de fase de una placa que contiene hiPSCs rodeadas por los fibroblastos. Las células se cultivaron durante 21 días en hES medios. (B, C) La misma colonia hiPSC después de la eliminación de la capa de fibroblastos circundante. (D) correctamente reprogramadas colonias hiPSC se identifican mediante tinción vivo con anticuerpos marcador Tra-1-81 superficie, cortar utilizando una aguja estéril (E, F) y se transfirieron a pocillos separados de una placa de 24 pocillos. <img alt="Figura 3" src="/files/ftp_upload/3416/3416fig3.jpg" /> Figura 3. La expresión de los marcadores de pluripotencia específicos Oct3 / 4, Nanog, Sox2, SSEA4 y Tra-1-60 en hiPSCs se determinó por inmunocitoquímica.

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Los autores no tienen nada que revelar.

El estudio de enfermedades humanas, especialmente neurológicas y neurodegenerativas, ha sido particularmente difícil debido a la inaccesibilidad de los adecuados modelos celulares humanos. La posibilidad de reprogramar células somáticas que se obtienen fácilmente en células madre pluripotentes inducidas y el potencial para diferenciarse en diversos tipos de células se abrió la posibilidad de crear modelos celulares de enfermedades genéticas. Además, iPSCs celebrar una gran promesa para el futuro de la medicina...

<table border="1"><tbody><tr><td> Reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td></tr><tr><td> KSR </td><td> Invitrogen </td><td> 10828 </td></tr><tr><td> Aminoácidos no esenciales </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Butirato sódico </td><td> Sigma </td><td> B5887 </td></tr><tr><td> Y27632 </td><td> Stemgent </td><td> 04-0012 </td></tr><tr><td> bFGF </td><td> Stemgent </td><td> 03-0002 </td></tr><tr><td> Tra-1-81 anticuerpo </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> Oct3 / 4 de anticuerpos </td><td> Santa Cruz </td><td> SC-8628 </td></tr><tr><td> Nanog anticuerpos </td><td> Señalización Celular Tecnología </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60 anticuerpo </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 anticuerpos </td><td> Señalización Celular Tecnología </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore </td><td> MAB4304 </td></tr><tr><td> CF1 MEFs </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td> Marcador de objetivo </td><td> Nikon </td><td> MBW10010 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr><tr><td> mTeSR1 </td><td> StemCell Tecnologías </td><td> 05850 </td></tr><tr><td> β-mercaptoetanol </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> polibreno </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Objeto marcador </td><td> Nikon </td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

El presente artículo presenta un protocolo de reprogramar los fibroblastos humanos adultos en las células madre pluripotentes inducidas (hiPSC) utilizando vectores retrovirales que codifican Oct3 / 4, Sox2, Klf4 y c-myc (OSKM) en presencia de sodio 1.3 butirato. Hemos utilizado este método para reprogramar el paso tardío (&gt; p10) fibroblastos humanos adultas derivadas de los pacientes la ataxia de Friedreich (GM03665, Coriell repositorio). El enfoque de reprogramación incluye protocolo de transducción de alta eficiencia utilizando centrifugación repetitiva de los fibroblastos en la presencia de virus que contienen medios de comunicación. Las colonias hiPSC reprogramadas se identificaron utilizando inmunotinción vivo para Tra-1-81, un marcador de superficie de las células pluripotentes, separado de los fibroblastos no reprogramadas y manualmente passaged 4,5. Estos hiPSC fueron trasladados a las placas de Matrigel y crecido en condiciones libres de alimentador, directamente desde la placa de la reprogramación. A partir de el primer paso, las colonias hiPSC demuestran características HES-lmorfología de Ike. Utilizando este protocolo más del 70% de las colonias seleccionadas pueden ser ampliado con éxito y se estableció en líneas celulares. Las líneas establecidas hiPSC muestra características marcadores de pluripotencia, incluyendo los marcadores de superficie TRA-1-60-4 y SSEA, así como los marcadores nucleares Oct3 / 4, Sox2 y Nanog. El protocolo que aquí se presenta se ha establecido y probado usando fibroblastos adultos obtenidos de pacientes con AF e individuos de control 6, fibroblastos humanos recién nacidos, así como los queratinocitos humanos.

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

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

Se presenta un protocolo para la reprogramación eficiente de las células somáticas humanas en células madre pluripotentes inducidas (hiPSC) utilizando vectores retrovirales que codifican Oct3 / 4, Sox2, Klf4 y c-myc (OSKM) y la identificación de hiPSC correctamente reprogramado por tinción en vivo con Tra- 1-81 anticuerpo.

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 ...

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19.8K vistas.  University of Texas M.D. Anderson Cancer Center. Se presenta un protocolo para la reprogramación eficiente de las células somáticas humanas en células madre pluripotentes inducidas (hiPSC) utilizando vectores retrovirales que codifican Oct3 / 4, Sox2, Klf4 y c-myc (OSKM) y la identificación de hiPSC correctamente reprogramado por tinción en vivo con Tra- 1-81 anticuerpo.

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

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 ...

Investigación

Biología

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

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 ...

Uso de biopsias de piel para generar podocitos derivados de pacientes

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Autor destacado: Generación de podocitos derivados de pacientes a partir de biopsias de piel  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Generación de podocitos derivados de pacientes a partir de biopsias de piel

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...