Este trabajo fue financiado por la Sociedad Max Planck.

1. Preparación de Matrigel recubiertos con placas y los medios <ol><li> Preparación de Matrigel recubierto con Matrigel placas se ha encontrado que es un sustrato preferido para la unión de diferenciar EBs y también promueve la excrecencia eficiente de las neuronas de estos 10. <ol><li> Descongelar 10 ml de noche en hielo Matrigel. </li><li> Al día siguiente, se diluye la Matrigel gelatinosa con dos volúmenes de hielo frío DMEM/F-12 y preparar partes alícuotas de 1 ml en tubos de 15 ml preenfriados cónicos que pueden almacenarse congeladas a -20 ° C. </li><li> Decidir sobre un formato de placa para la diferenciación neuronal. Por ejemplo, como punto de partida, se recomienda llevar a cabo una ronda inicial de la diferenciación en un formato de 12-así. Pre-enfriar cuatro placas de 12 pocillos a 4 ° C. Disolver una alícuota de Matrigel congelado mediante la adición de 9 ml de pre-refrigerado DMEM/F-12 seguido por pipeteando arriba y abajo hasta que todo el Matrigel prediluido ha descongelado y se disolvió. </li><li> Entonces, Transferir el contenido en un tubo de 50 ml nuevo que contiene 15 ml de DMEM/F-12 en hielo (dilución final Matrigel: 1:75). A continuación, añadir 0,5 ml de Matrigel diluido a cada pocillo de las pre-enfriados 12-pocillos. Envuelva las placas con Parafilm y se deja reposar durante la noche a temperatura ambiente. </li><li> Al día siguiente, transferir las placas a 4 ° C. Las placas recubiertas se pueden almacenar durante varias semanas antes de su uso. </li></ol></li><li> Medios de comunicación </li></ol> EB medio de formación (~ 15 ml necesario para realizar la diferenciación en una placa de 96 pocillos - ver Tabla 1 y el esquema en la figura 2): DMEM/F-12 con 0,4% (PVA) 1x N2 1x B27 0,05% de BSA 20 ng / ml FGF2 10 mM Y-27632 1x PenStrepGln Medio de diferenciación (~ 30 ml necesario para realizar la diferenciación en una placa de 96-pocillos, seguido por la formación de neuronas en una Matrigel recubierto de 12-pocillos, véase la Tabla 1): DMEM/F-12 1x N2 <br/&gt; 1x B27 0,05% de BSA 0,5 mM PD0325901 15 SB431542 mu M 0,5 mM Dorsomorphin 1x PenStrepGln 2. EB forzado Formación <ol><li> Crecer hPSCs bajo tus preferidos sin conexión en condiciones. Utilizamos MEF medio acondicionado en Matrigel revestidos 6-y las placas 15. Sin embargo, otros sistemas de cultivo, como medio definido caseros o comerciales también debería funcionar. En el momento de iniciar un experimento diferenciación, hPSCs debe ser sub-confluente y creciendo activamente. </li><li> Mecánicamente eliminar espontáneamente colonias diferenciadas usando una punta de pipeta de plástico estéril bajo un microscopio estéreo. </li><li> Lávese las restantes colonias indiferenciadas con PBS, y se resumen a células individuales utilizando Accutase contiene 1X Y-27632. Pellets individuales células por centrifugación a 200 xg durante 2 min, se resuspende en un pequeño volumen de medio de formación de EB y determinar título celular utilizando un hematocitómetro. </li><li> Añadir una alícuota de células ael EB restante medio de formación para dar como resultado un título entre 40.000 y 80.000 células por ml. </li><li> Usando una pipeta multicanal, transferir 100 l por pocillo a una placa de fondo 96V, resultando en 4.000 a 8.000 células por pocillo. Centrifugación placa de 96 pocillos en una centrífuga de plato oscilante a 400 xg durante 1 min para recoger las células en la parte inferior de los pocillos, y transferir a la incubadora de cultivo celular. EBS se forman durante la noche, como se muestra en la figura 1. </li></ol> 3. Neuroectodermo Inducción <ol><li> Al día siguiente (día 0), recoger EBs de 96V de la placa bajo un microscopio estéreo y transferencia en un pequeño volumen en una placa de 3,5 cm bacteriano que contiene 2 ml de medio de diferenciación. Agitar suavemente el plato durante unos segundos para lavar el EBS. </li><li> Añadir 100 l de medio de diferenciación por pocillo de un ultra-bajo-apego fondo en U de 96 pocillos. Bajo un microscopio estereoscópico, la transferencia de EBS en pequeños volúmenes del lavado de platos en el 96U-pozos (uno por EB quell). Place 96U placa en cultivo celular incubadora durante 4 días para permitir la formación neuroectodermo. </li></ol> 4. Neurona Formación <ol><li> El día 4, preparar un plato de lavado como en el paso 3,1 y adicionalmente sustituir DMEM/F-12 en un pre-calentado Matrigel recubierto 12-así placa por medio de diferenciación (1 ml por pocillo). </li><li> Revestimiento de EBs <ol><li> Recoger EBs de placa de 96U, lavar estas semillas como anteriormente, y con cuidado uno por uno en los pocillos de la Matrigel recubierto de 12-pocillos (aproximadamente 8 CE por pocillo): EBS se distribuirá uniformemente dentro de los pozos para permitir el crecimiento sin perturbaciones de neuronas en los próximos días (ver &quot;día 5&quot; imagen de la Figura 2). </li><li> Antes de la transferencia de la placa de respaldo 12-bien en la incubadora, permitir EBS para acoplar con flojedad de alrededor de 10 min a TA. Luego, transferir cuidadosamente 12-así placa de cultivo celular incubadora. Las neuronas se crecen desde el chapado de EBs de forma radial dentro de los próximos 4 días, como shown en la Figura 2 (&quot;Día 8&quot; La imagen de la derecha). </li></ol></li></ol> 5. Neurona Formación como monocapas <ol><li> Como alternativa a los pasos del punto 4), en el día 4 del procedimiento, recoger EBs de en una placa de 3,5 cm bacteriano que contiene 2 ml de medio de diferenciación, a continuación, transferir embrioides en un pequeño volumen a un plato que contiene PBS, y luego en otro plato que contiene Accutase. </li><li> Deje digerir a células individuales, se centrifuga a 200 xg durante 2 min, se resuspende en un pequeño volumen de medio de diferenciación y determinar título celular utilizando un hematocitómetro. Ajuste título a 1-2x10 6 células por ml usando medio de diferenciación (sin S-27632). </li><li> Células de la placa hacia fuera en pre-calentado Matrigel recubierto de 12-pocillos (1 ml por pocillo), y la transferencia a la incubadora de cultivo celular. Formación de las neuronas como monocapas se observó entre los días 7 a 8 (Figura 3C). Cabe señalar, sin embargo, que esta variante de monocapael procedimiento no está totalmente optimizado con respecto a la supervivencia de las células y el substrato más adecuado. </li></ol>

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La mayoría de los protocolos de diferenciación que implican la formación de EB hPSCs, incluyendo nuestro procedimiento previamente publicado 10, se basa en el manual o con ayuda de enzima de recolección hESCs como agregados, lo que inevitablemente conduce a la heterogeneidad en los tamaños de EB. Este hecho conduce a variabilidad en la eficacia con la diferenciación de algunas líneas celulares, haciendo así difícil análisis sistemáticos, en particular, a una escala de rendimiento medio. Además, la...

hPSCs, que comprende embrión derivado de células madre embrionarias humanas (hESCs) y células madre pluripotentes inducidas (hiPSCs), son pluripotentes sentido de que pueden dar lugar a diversos linajes celulares in vitro 1-2. Numerosos tipos de células diferenciadas se han derivado de hESCs hasta la fecha, apoyando la idea de que estas células presentan valiosas herramientas para la investigación científica y basados ​​en células enfoques de cribado, y mantienen la promesa para células basadas en la terapéutica. Protocolos de diferenciación neuronal para hESCs son generalmente complejas y requiere mucho tiempo, ya que a menudo se basan en la inducción de primera general destino neural, seguido de aislamiento manual de los progenitores neuronales, y la diferenciación terminal posterior en un tipo de neurona dada de interés 3-6. En algunos respecto, esta complejidad no es sorprendente e inevitable dado que tal estado-de-la-arte dirigido protocolos de diferenciación por etapas tienden a imitar el desarrollo temprano humano, which es en sí mismo muy complejo. Por otra parte, puede, en parte, también reflejan la falta de entendimiento de los procesos moleculares subyacentes, lo que resulta en protocolos de diferenciación que aún están lejos de ser totalmente optimizado. Con respecto a la conversión de hPSCs indiferenciadas en neuroectodermo, Pera et al. encontraron que la supresión de BMP / SMAD1/5/8 señalización aumenta la inducción neural de hESCs 7. Además, la inactivación de Smad2 / 3 de señalización se ha demostrado para promover la formación neuroectodermo 8. Por consiguiente, la inactivación combinada de estas dos vías tiende a conducir a la inducción neural más eficiente de hPSCs 4,9. Más recientemente, se ha demostrado que una tercera vía de señalización, FGF / ERK, sirve como un represor fuerte de los primeros pasos de compromiso neuroectodérmico en hESCs. Por el contrario, la represión simultánea de estos tres caminos conducen a casi homogéneo conversión de hESCs en neuroectodermo conen tan sólo cuatro días 10. Posteriormente, la diferenciación terminal altamente eficiente en neuronas funcionales se observó dentro de ocho días. Las neuronas obtenidas fueron probablemente las neuronas sensoriales del sistema nervioso periférico, lo que puede explicar en parte su derivación rápida 10. Errores de mantenimiento neurona sensorial se considera una causa de ciertos trastornos humanos, tales como la disautonomía familiar 11. Para el modelado de dichas enfermedades basadas en la tecnología hiPSC 12, para la caracterización funcional adicional, así como para fines aplicados no requieren ningún tipo particular de neuronas, este procedimiento de diferenciación puede presentar una base útil. Sin embargo, la estrategia de diferenciación de lo que originalmente se empleó la formación de los cuerpos involucrados embrionarias (EBS) de grupos de 10 colonias hESC, y esto dio lugar a un buen grado de heterogeneidad en cuanto a tamaños de EB, y también parecía poner en peligro la eficiencia de formación de neuronas en algunas celíneas ll. Además, debido a la heterogeneidad en los tamaños EB, la capacidad para probar sistemáticamente los efectos de factores de crecimiento adicionales o moléculas pequeñas durante y después de la formación de neuronas parecían ser algo confundido. En este informe, hemos adaptado nuestro procedimiento anterior para un rendimiento medio-compatible, forzado agregación basado en la técnica para generar EBS en un tamaño muy controlado de manera que también se observan en otros contextos 13. Posteriormente, el EBS generada en forma de V pocillos de placas de 96 pocillos se transfirieron a en forma de U ultra-bajo fijación placas 96-pocillos, para iniciar la formación neuroectodermo en suspensión. Cuatro días más tarde, el EBS se podría sembraron dar lugar a neuronas terminalmente diferenciadas con alta eficiencia y homogeneidad. Alternativamente, día-4 EBS se disocia en células individuales y se colocaron como tal, lo que resultó en monocapas de baja densidad de neuronas humanas. Resultados fueron coherentes con los obtenidos hasta el momento dos de forma independientelíneas de células madre, Rived HuES6 y NCl3. Como un requisito previo, la formación de EB éxito era estrictamente dependiente de la utilización de un polímero sintético, alcohol de polivinilo (PVA), junto con inhibidor de la ROCA Y-27632 conocido para promover la supervivencia hESC 13-14. Como resultado de ello, la homogeneidad de la EBS formado en el 96-V-placas se mejoró considerablemente en comparación a la formación de EBs como cultivos en masa basado en técnicas de splitting aleatorios. Así, este sistema proporciona una plataforma significativamente mejorada para la formación de neuroectodermo en cultivo en suspensión, seguido por la formación de neuronas altamente controlada en condiciones adherentes.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Matrigel HC </td><td> Becton Dickinson </td><td> 354263 </td><td> Ver texto para más detalles sobre el manejo de </td></tr><tr><td> DMEM/F-12 </td><td> Life Technologies </td><td> 21331-020 </td><td></td></tr><tr><td> Poli (alcohol vinílico) </td><td> Sigma </td><td> 363170 </td><td> Disolver en 0,4% en DMEM/F-12 utilizando un baño de agua ultrasónico / evitar el sobrecalentamiento / puede mantenerse a 4 ° C durante varias semanas </td></tr><tr><td> Suplemento N2 </td><td> Life Technologies </td><td> 17502-048 </td><td> 100X, almacenar como alícuotas congeladas </td></tr><tr><td> B27 Suplemento </td><td> Life Technologies </td><td> 12587-010 </td><td> Tienda 50X, como alícuotas congeladas </td></tr><tr><td> Albúmina de suero bovino </td><td> Sigma </td><td> A1595 </td><td> 10% = 500X, almacenar en alícuotas congeladas </td></tr><tr><td> L-Glutamina con penicilina / estreptomicina </td><td> PAA </td><td> P11-013 </td><td> O el equivalente </td></tr><tr><td> FGF2 </td><td> Peprotech </td><td> 100-18B </td><td> Disolver en 10 ug / ml en 0,1% de BSA / PBS = 1000X tienda, como alícuotas congeladas </td></tr><tr><td> ROCA inhibidor Y-27632 </td><td> abcamBiochemicals </td><td> Asc-129 </td><td> Disolver en 10 mM en DMSO = 1000X tienda, como alícuotas congeladas </td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td> O el equivalente </td></tr><tr><td> Accutase </td><td> PAA </td><td> L11-007 </td><td></td></tr><tr><td> 96-y de fondo en V placas </td><td> Nunc </td><td> 277143 </td><td></td></tr><tr><td> PD0325901 </td><td> Axon MedChem </td><td> Axon 1408 </td><td> Disolver en 0,5 mM en DMSO = 1000X tienda, como alícuotas congeladas </td></tr><tr><td> SB431542 </td><td> abcamBiochemicals </td><td> Asc-163 </td><td> se disuelven a 15 mM en DMSO = 1000X tienda, como alícuotas congeladas </td></tr><tr><td> Dorsomorphin </td><td> Santa Cruz </td><td> sc-200689 </td><td> disolver en 0,5 mM en DMSO = 1000X tienda, como alícuotas congeladas </td></tr><tr><td> U de 96 pocillos de fondo ultra bajo placas de fijación </td><td> Corning </td><td> 7007 </td><td></td></tr><tr><td> beta-tubulina III anticuerpos (ratón) </td><td> Sigma </td><td> T8660 </td><td> utilizar a 1:1000 </td></tr><tr><td> beta-tubulina III anticuerpos (conejo) </td><td> Covance </td><td> PRB-435P </td><td> utilizar a 1:2000 </td></tr><tr><td> Brn3a (POU4F1) de anticuerpos </td><td> Santa Cruz </td><td> sc-8429 </td><td> utilizar a 1:500 </td></tr><tr><td></td><td></td><td></td><td> Tabla 1. &lt;/ Strong&gt; reactivos y equipos específicos. </td></tr></tbody></table>

rapid and efficient generation of neurons from human pluripotent stem cells in a multititre plate format

Los protocolos existentes para la generación de neuronas a partir de células madre pluripotentes (hPSCs) suele ser tedioso, ya que son procedimientos de múltiples etapas que implica el aislamiento y la expansión de las células precursoras neurales, antes de la diferenciación terminal. En comparación con estos métodos consumen mucho tiempo, recientemente se ha encontrado que la inhibición combinada de tres vías de señalización, TGFβ/SMAD2, BMP/SMAD1, FGF y / ERK, promueve la rápida inducción de la neuroectodermo de hPSCs, seguido de inmediato diferenciación en neuronas funcionales. Aquí, hemos adaptado nuestro procedimiento para un formato de placa novela multititre, para mejorar aún más su reproducibilidad y para que sea compatible con mediados de rendimiento de las aplicaciones. Se compone de cuatro días de formación neuroectodermo en esferas flotantes (cuerpos embrioides), seguido por un período de cuatro días de diferenciación en neuronas en condiciones adherentes. La mayoría de las células obtenidas con este protocolo parecen ser las neuronas sensoriales bipolares. Por otra parte,el procedimiento es altamente eficaz, no requiere habilidades particulares de expertos, y se basa en un simple medio químicamente definido con rentables moléculas pequeñas. Debido a estas características, el procedimiento puede servir como una plataforma útil para la investigación funcional adicional, así como para el cribado basado en células humanas se aproxima requiere neuronas sensoriales o neuronas de cualquier tipo.

En nuestro informe anterior, en línea con muchos otros, EBS desde hPSCs podría generarse simplemente replating agregados sobre la división de rutina de hPSCs en platos de baja fijación 10. Esto usualmente resulta en una amplia distribución de tamaños de EB resultantes (Figura 1 C, arriba). Otro problema que resulta de esto es que EBS mantienen en suspensión tienden a agregarse entre sí, dando lugar a una heterogeneidad aún mayor de tamaños de EB. Para evitar estos problemas, por lo ...

Watch this Scientific Journal Video about Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format at JoVE.com

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

Protocolos para la diferenciación neuronal de las células madre humanas pluripotentes (hPSCs) son a menudo mucho tiempo y requieren considerables conocimientos de cultivo de células. Aquí, hemos adaptado una molécula pequeña de un procedimiento basado en una diferenciación a un formato de placa de multititre, que permite la generación sencilla, rápida, y eficiente de las neuronas humanas de una manera controlada.

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 ...

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43.5K vistas.  Max Planck Institute for Molecular Biomedicine. Protocolos para la diferenciación neuronal de las células madre humanas pluripotentes (hPSCs) son a menudo mucho tiempo y requieren considerables conocimientos de cultivo de células. Aquí, hemos adaptado una molécula pequeña de un procedimiento basado en una diferenciación a un formato de placa de multititre, que permite la generación sencilla, rápida, y eficiente de las neuronas humanas de una manera controlada.

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

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 ...

Investigación

Biología

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Rápido y Eficiente de pez cebra Genotipado Utilizando PCR con análisis del derretimiento de alta resolución

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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 ...

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

La esporulación eficiente de<em&gt; Saccharomyces cerevisiae</em&gt; En un formato de 96 multipocillos

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

Generación eficiente y consistente de epitelio pigmentario retiniano/cordoides de montaje plano a partir de ojos humanos para análisis histológico

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...