Gracias a Tim Doyle, PhD y el Centro Stanford de imágenes in vivo para obtener ayuda con imágenes de bioluminiscencia. También gracias a Ngan Huang, PhD para compartir su técnica de células madre de la co-inyección con una solución de la matriz. Por último, gracias a Steve Felt, Ph.D. para obtener ayuda con el cuidado de animales veterinarios.

<ol><li> Construcción de doble reportero Gene fusión <ol><li> Con el fin de realizar imágenes de bioluminiscencia de células madre embrionarias humanas, primero tiene que obtener las células que expresan establemente un gen reportero de la luciferasa como luciferasa de luciérnaga impulsado por un promotor constitutivo como ubiquitina o EF1A. </li><li> El objetivo de este protocolo es el gen reportero de aplicaciones, por lo que los procedimientos detallados que no se proporcionan aquí. Sin embargo, la estrategia general de nuestro laboratorio es el uso de un vector de construcción de fusión doble que contiene luciferasa de luciérnaga (fluctuaciones) y aumento de proteína verde fluorescente (EGFP) separadas por un espaciador en pCDNA 3.1 +. </li><li> En pocas palabras, nuestro gen de fusión doble se encontraba originalmente aguas abajo del promotor de citomegalovirus en pcDNA 3.1 (+), por lo que el fragmento de 3,3 kpb fue eliminado mediante la digestión NdeI y NotI y contundente final ligó en el sitio de clonación múltiple de una auto-inactivar (SIN ) vector lentiviral, bajo control de un promotor de la ubiquitina. </li></ol></li><li> Transducción lentiviral <ol><li> hESCs pueden ser transducidas 3-5 días después de la aprobación a una multiplicidad de infección (MOI) de 10 (título viral de aproximadamente 107 células se incubaron con 106). </li><li> La acción viral descongelado se puede agregar directamente a los medios de células madre nuevas. </li><li> Actualizar los medios de comunicación 12 y 24 horas más tarde. </li><li> Después de 48 h, la eficiencia de transducción puede por evaluadas cualitativamente mediante microscopía de fluorescencia. Posteriormente, FACS se puede utilizar para aislar las células infectadas. </li></ol></li><li> HESCs cultivo y la introducción de imágenes de Set-up <ol><li> La cultura de su hESCs positivo luciferasa en el alimentador sin condiciones. Por lo general siguen el estándar de protocolo de WiCell y el uso de placas de 6 pocillos recubiertas de factor de crecimiento reducido Matrigel, utilizando los medios de comunicación acondicionado o comercial, libre de enlace de los medios de comunicación. </li><li> Para detectar las células positivas luciferasa, usted tendrá que tener la sonda reportero D-luciferina ya preparados. Para ello luciferina alícuota, en los preparativos 1-1.5 mL a una concentración final de 45 mg / ml. Mantener las alícuotas a -20 ° C cuando no estén en uso y evitar la exposición a la luz cubriendo con una toalla de papel, cuando no en el almacenamiento. </li><li> Con el fin de visualizar las células positivas luciferasa, usamos IVIS 50 y 200 sistemas de imágenes IVIS (Xenogen Corporation, Alameda, CA). Este último sistema incluye un aparato integrado isoflourane y cámara de inducción de la anestesia temporal de animales pequeños. </li></ol></li><li> En las imágenes de bioluminiscencia in vitro de hESCs <ol><li> En imágenes de bioluminiscencia in vitro, es importante mantener condiciones de esterilización. Por lo tanto, el sistema de imágenes deben ser estériles, de preferencia en una sala de cultivo celular. </li><li> Antes de imágenes, eliminar los medios de comunicación celular y añadir lo suficiente para cubrir PBS las células. Por ejemplo, añada 1 ml de PBS en cada pocillo de una placa de 6 pocillos que contienen células madre de sus culturas. </li><li> La proporción de D-luciferina de PBS debe ser 1:100, por lo que añadir 10 l de D-luciferina descongelado a cada pocillo de la placa de 6 pocillos. </li><li> Espere un minuto, luego tomar una imagen con un tiempo de exposición de 1 segundo. Si la señal es débil, aumentar el intervalo entre la exposición y vuelva a intentarlo. </li><li> La señal bioluminiscente refleja el número de células, por lo que los ensayos de cuantificación se puede realizar la señal que se correlacionan con el número de células diferentes. </li></ol></li><li> Preparación de células de ratón y la inyección de imágenes in vivo <ol><li> Gen reportero de imagen proporciona un método de alto rendimiento, bajo costo y sensible para el seguimiento de las células después del trasplante durante días, semanas e incluso meses. Esto es especialmente importante, ya que las células madre trasplantadas a menudo forman teratomas, son rechazados por el sistema inmune del huésped, o simplemente morir. </li><li> El método más simple para la formación de teratoma de imágenes in vivo, es la inyección hESCs que expresan el gen reportero de la luciferasa de luciérnaga en la espalda de ratones SCID y adquirir imágenes bioluminiscentes con el sistema de IVIS. </li><li> Cuando esté listo para el trasplante de las células, el uso dispasa o colagenasa para aflojar las células, lavar varias veces, y suspender en una mezcla 1:1 del factor de crecimiento reducido matrigel y DMEM. Por lo general lo primero que mezclar las células con DMEM fría y luego añadir en el matrigel. Para cada sitio de la inyección subcutánea, se inyecta 200.000 células suspendidas en un volumen de 50 a 100 ul de la mezcla de matrigel / DMEM. El número de células se pueden ajustar en función de su aplicación. Recuerde que debe mantener todo en hielo antes de la inyección. </li><li> Una vez que las células están listas, anestesiar el ratón, colocándolo en la caja de la inducción con isoflurano flujo de encendido. Después de 1-5 minutos, el ratón está dormido y se puede colocar en la mesa de operaciones continuas con isoflurano. </li><li> Porque la piel natural de auto-fluorescencia y pueden oscurecer la imagen bioluminiscente, que tendrá que eliminar el vello de la cara posterior del ratón. La forma más sencilla de hacer esto es utilizar una máquina de afeitar eléctrica, pero también puede utilizar la depilación-geles. Limpie con alcohol para esterilizarla piel después </li><li> A continuación, establecer la suspensión de su celda en una jeringa. 23 a 27 agujas de calibre mejor trabajo, ya que no va a tapar con las células. Si la aguja es demasiado grande (&lt;calibre 23), el trayecto de la aguja puede dejar un gran agujero a través del cual las células pueden tener fugas de vuelta. </li><li> Inyectar las células en la piel en la espalda del ratón. Porque la piel es muy floja, con el pulgar e índice para pellizcar y estirar la zona que desee inyectar. Se inyecta bajo la piel, teniendo cuidado de no perforar demasiado profundamente. Además, trate de mantener la aguja se deslice fuera del sitio de la inyección, mientras que el émbolo: esto evitará la creación de un agujero en la piel a través de la cual las células pueden tener fugas si se intenta inyectar de nuevo. </li><li> Después que las células se inyectan, esperar unas horas para permitir que el ratón para despertar y empezar a correr antes de volver a anestesiar a imágenes de bioluminiscencia. Si lo hace, evita la toxicidad isoflurano. </li></ol></li><li> En imágenes de bioluminiscencia in vivo de las células trasplantadas <ol><li> Para obtener imágenes de toda la bioluminiscencia animal, hacemos una inyección intraperitoneal de D-luciferina a 375 peso mg / kg. Pesar al animal para calcular la dosis antes de la inyección. </li><li> Inyectar el D-luciferina en el peritoneo, justo al lado de la línea media. Tenga cuidado de no ir demasiado profundo o puede dañar los órganos internos. Si el ratón comienza a despertar, volver a meterla en la caja de octavos de final y esperar un minuto o dos. En los ratones, la bioluminiscencia máximo ocurre generalmente 15-40 minutos después de la inyección. </li><li> Recuerde que debe colocar el papel negro mate en el cuadro de imagen para ayudar a absorber cualquier luz no emitida por el hESCs. Coloque el ratón (hasta parte trasera) en la cámara de imágenes con isoflurano, en la parte superior del papel negro. Comience con un tiempo de exposición de 10 segundos. Si la señal está saturada, pruebe a reducir el tiempo de exposición, si es demasiado débil, incremento de la exposición. </li><li> Una vez que el tiempo de exposición de la señal ha sido optimizado para el ratón, comenzar a tomar imágenes cada minuto hasta que la señal llega a un máximo. Esto se logra mejor utilizando el software de imágenes para seleccionar &quot;regiones de interés (ROI)&quot;, que cubren la zona de inyección. Por el control de la intensidad de la señal en cada retorno de la inversión puede determinar con facilidad cuando la señal empieza a disminuir, lo que indica que la intensidad de la señal ha alcanzado el máximo. La máxima de la señal debe ser utilizado como sus datos finales. </li><li> Cuando estés satisfecho con tus imágenes, eliminar el ratón de isoflurano y deje que se despierta en su jaula. Por lo general, el ratón debe despertar dentro de 15 minutos. </li><li> Reportero de la bioluminiscencia de imágenes de genes puede repetirse a diario, semanal y mensual durante el tiempo que el hESCs sobrevivir en el animal. Señal de un teratoma en desarrollo aumentará de forma exponencial con el tiempo, típicamente del orden de semanas, ya que el hESCs empiezan a proliferar. </li><li> Después de la evolución en el tiempo deseado de imágenes de bioluminiscencia se adquiere, el animal puede ser sacrificado, y las secciones de tejido utilizado para el estudio histológico. </li></ol></li></ol>

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En comparación con otras modalidades como la PET y la RM, la bioluminiscencia tiene una resolución espacial limitada y penetración en los tejidos debido a la reducción de la energía relativamente débil de los fotones emitidos (2-3 eV), por estas razones que hasta ahora no ha sido aplicable en los animales grandes. Sin embargo, la bioluminiscencia tiene la ventaja de ser de bajo costo, alto rendimiento, y no invasiva, por lo que es muy conveniente que en las células madre in vivo de seguimiento en pequeños animales. Bioluminiscencia ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>BD Matrigel&#x2122; Basement Membrane Matrix</td>
<td>Growth factor reduced (optional: phenol-red free)</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-Luciferin Firefly, potassium salt</td>
<td>&nbsp;</td>
<td>Biosynth AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td>
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<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>6-well tissue culture-treated plates</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>92006</td>
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in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

El descubrimiento de células madre embrionarias humanas (hESCs) se ha incrementado dramáticamente las herramientas disponibles para los científicos médicos interesados ​​en la medicina regenerativa. Sin embargo, la inyección directa de hESCs, y las células diferenciadas a partir de hESCs, en los organismos vivos hasta el momento ha sido obstaculizada por la muerte celular significativa, la formación de teratomas, y el rechazo inmune del huésped. Entender el comportamiento de células madre in vivo después de un trasplante requiere nuevas técnicas de imagen para controlar la localización longitudinal células madre, la proliferación y viabilidad. Imagen molecular ha dado a los investigadores un medio de alto rendimiento, bajo costo y sensible para el seguimiento de la proliferación celular in vivo durante días, semanas e incluso meses. Este avance se ha incrementado significativamente la comprensión de la cinética de espacio-temporal de injerto células madre, proliferación y formación de teratoma en sujetos vivos. Un avance importante en la proyección de imagen molecular ha sido la extensión de ensayos de gen reportero no invasivo de la biología molecular y celular en las plataformas de imágenes en vivo en varias modalidades. Estos genes reporteros, bajo el control de promotores y potenciadores de ingeniería que se aprovechan de la maquinaria transcripcional de la célula huésped s, se introducen en las células usando una variedad de métodos vectoriales y no vectoriales. Una vez en la célula, los genes indicadores pueden ser transcritos o bien constitutivamente o sólo en determinadas condiciones biológicas o celular, dependiendo del tipo de promotor utilizado. Transcripción y traducción de genes reporteros en proteínas bioactivas es detectado con instrumentos sensibles, no invasivo (por ejemplo, cámaras CCD) con la generación de la señal de las sondas, tales como D-luciferina. Para evitar la necesidad de luz de excitación para realizar un seguimiento in vivo de células madre que se requiere para imágenes de fluorescencia, bioluminiscencia reportero de sistemas de imágenes gen sólo requieren una investigación administrada de forma exógena para inducir la emisión de luz. Luciferasa de luciérnaga, derivado de la luciérnaga pyralis Fotino, codifica una enzima que cataliza la D-luciferina a la ópticamente activos oxyluciferin metabolito. Actividad óptica puede ser controlado con una cámara CCD externa. Establemente células transducidas que llevan el reportero construir dentro de su ADN cromosómico pasará el reportero construcción de ADN a las células hijas, permitiendo un seguimiento longitudinal de la supervivencia y la proliferación de células madre in vivo. Por otra parte, porque la expresión del producto del gen reportero se requiere para la generación de señales, las células madre y la hija única solución viable a crear la señal de la bioluminiscencia, la apoptosis o muerte no. En este video, los materiales específicos y los métodos necesarios para la proliferación de células madre y el seguimiento de la formación de teratomas con imágenes de bioluminiscencia se describen.

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Con el creciente interés en terapias con células madre, las técnicas de imagen molecular son ideales para el comportamiento de las células madre de seguimiento tras el trasplante. Genes luciferase reportero le han permitido no invasiva, la evaluación repetida de la supervivencia celular, la ubicación y la proliferación in vivo. Este vídeo demuestra cómo realizar el seguimiento proliferación de células madre en un ratón vivo.

In vitro e in vivo de imágenes de bioluminiscencia gen reportero de células estaminales embrionarias humanas

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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Research

JoVE Journal

Biology

21.7K vistas.  Stanford University School of Medicine. Con el creciente interés en terapias con células madre, las técnicas de imagen molecular son ideales para el comportamiento de las células madre de seguimiento tras el trasplante. Genes luciferase reportero le han permitido no invasiva, la evaluación repetida de la supervivencia celular, la ubicación y la proliferación in vivo. Este vídeo demuestra cómo realizar el seguimiento proliferación de células madre en un ratón vivo.

Video: In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivación de células estaminales embrionarias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Investigación

Biología

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondeo de la actividad mitocondrial complejo en células estaminales embrionarias humanas

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

En el etiquetado in vitro de células estaminales embrionarias humanas de Resonancia Magnética

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

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

Culture and Maintenance of Human Embryonic Stem Cells

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

La cultura y el mantenimiento de células estaminales embrionarias humanas

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

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelación y descongelación células estaminales embrionarias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análisis de Crecimiento Humano Colonia células madre embrionarias

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...