Merci à Tim Doyle, PhD, et le Centre Stanford pour l&#39;imagerie in vivo pour l&#39;aide à l&#39;imagerie par bioluminescence. Merci aussi à Ngan Huang, PhD pour le partage de sa technique sur les cellules souches co-injection avec une solution de la matrice. Enfin, grâce à Steve Felt, Ph.D. pour l&#39;aide aux soins des animaux à usage vétérinaire.

<ol><li> Construction du gène de fusion Reporter double <ol><li> Afin d&#39;effectuer l&#39;imagerie par bioluminescence de cellules souches embryonnaires humaines, vous devez d&#39;abord obtenir des cellules expriment de façon stable un gène rapporteur luciférase tels que la luciférase de luciole piloté par un promoteur constitutif, comme l&#39;ubiquitine ou EF1a. </li><li> L&#39;objectif de ce protocole est sur les applications du gène rapporteur, alors procédures détaillées ne sont pas fournies ici. Cependant, la stratégie générale de notre laboratoire est d&#39;utiliser un vecteur double fusion qui contient de construire la luciférase de luciole (fluctuations) et amélioré la protéine fluorescente verte (EGFP) séparés par une entretoise au sein pCDNA 3.1 +. </li><li> En bref, notre gène double fusion était initialement situé en aval du promoteur de cytomégalovirus dans pcDNA 3.1 (+), de sorte que le fragment de 3,3 kpb a été excisé par digestion Ndel et Notl et à bout émoussé ligaturé dans le site de clonage multiple d&#39;une auto-inactivation (NAS ) vecteur lentiviral, sous contrôle d&#39;un promoteur de l&#39;ubiquitine. </li></ol></li><li> Transduction lentiviraux <ol><li> CSEh peuvent être transduites 3-5 jours après le passage à une multiplicité d&#39;infection (MOI) de 10 (titre viral d&#39;environ 107 106 cellules incubées avec). </li><li> Le stock dégelé virale peut être ajouté directement aux médias CSEh frais. </li><li> Actualiser les médias 12 et 24 heures plus tard. </li><li> Après 48 h, l&#39;efficacité de transduction peut par une évaluation qualitative en utilisant la microscopie à fluorescence. Par la suite, FACS peut être utilisé pour isoler les cellules infectées. </li></ol></li><li> CSEh Culture et Introduction à l&#39;imagerie Set-up <ol><li> Culture de votre luciférase CSEh positifs dans le chargeur sans conditions. Nous généralement suivre le protocole standard WiCell et l&#39;utilisation des plaques 6 puits recouvert de facteur de croissance réduite de Matrigel, en utilisant soit des milieux conditionnés ou commerciales, chargeur-média libre. </li><li> Pour détecter les cellules luciférase positif, vous aurez besoin d&#39;avoir la sonde journaliste de D-luciférine déjà préparé. Pour ce faire partie aliquote luciférine, en 1 à 1,5 ml des préparations à une concentration finale de 45 mg / mL. Gardez les aliquotes à -20 ° C lorsqu&#39;il n&#39;est pas utilisé et éviter l&#39;exposition à la lumière en couvrant avec un papier absorbant quand il n&#39;est pas dans le stockage. </li><li> Afin de visualiser les cellules luciférase positif, nous utilisons IVIS 50 et 200 systèmes d&#39;imagerie IVIS (Xenogen Corporation, Alameda, Californie). Ce dernier système comprend un appareil intégré et de la chambre d&#39;induction isoflourane pour l&#39;anesthésie temporaire de petits animaux. </li></ol></li><li> Dans l&#39;imagerie par bioluminescence in vitro des CSEh <ol><li> Pour l&#39;imagerie par bioluminescence in vitro, il est important de maintenir des conditions stériles. Par conséquent, le système d&#39;imagerie doivent être stériles, de préférence dans une salle de culture cellulaire. </li><li> Avant d&#39;imagerie, retirez le support de cellule et d&#39;ajouter juste assez pour couvrir PBS les cellules. Par exemple, ajouter 1 ml de PBS dans chaque puits d&#39;une plaque de 6 puits contenant vos cultures de CSEh. </li><li> Le rapport de la D-luciférine à PBS doit être 1:100, donc ajouter 10 ul de décongelés D-luciférine dans chaque puits de la plaque de 6 puits. </li><li> Attendez une minute, puis prendre une image en utilisant un temps d&#39;exposition de 1 seconde. Si le signal est faible, augmentez l&#39;intervalle d&#39;exposition et essayez à nouveau. </li><li> Le signal bioluminescent reflète le nombre de cellules, donc des tests de quantification peut être effectuée en corrélation du signal avec le nombre de cellules différentes. </li></ol></li><li> Préparation de la souris et les cellules d&#39;injection pour l&#39;imagerie in vivo <ol><li> Gène rapporteur imagerie fournit une méthode à haut débit, peu coûteuses et sensibles pour les cellules de suivi après la transplantation au fil des jours, des semaines, voire des mois. Ceci est particulièrement important car les cellules souches greffées forment fréquemment tératomes, sont rejetés par le système immunitaire de l&#39;hôte, ou tout simplement mourir. </li><li> La méthode la plus simple pour la formation de tératome imagerie in vivo consiste à injecter de CSEh qui expriment le gène rapporteur luciférase firefly dans le dos des souris SCID et d&#39;acquérir des images de bioluminescence avec le système IVIS. </li><li> Lorsque vous êtes prêt à transplanter des cellules, l&#39;utilisation ou la collagénase dispase à desserrer les cellules, se laver plusieurs fois, et les suspendre dans un mélange 1:1 de facteur de croissance réduit Matrigel et DMEM. Généralement nous avons d&#39;abord mélanger les cellules avec du DMEM et refroidi, puis ajouter dans le Matrigel. Pour chaque site d&#39;injection sous-cutanée, nous injectons 200 000 cellules en suspension dans un volume de 50 à 100 ul du mélange matrigel / DMEM. Le nombre de cellules peut être ajustée en fonction de votre application. N&#39;oubliez pas de garder tout sur la glace avant l&#39;injection. </li><li> Une fois que les cellules sont prêtes, anesthésier la souris en la plaçant dans la boîte à induction à débit isoflurane allumé. Après 1-5 minutes, la souris est endormi et peut être placé sur la table d&#39;opération avec l&#39;isoflurane continu. </li><li> Parce que la fourrure naturelle d&#39;auto-fluorescence et peuvent obscurcir l&#39;image bioluminescents, nous aurons besoin pour enlever les poils de la face arrière de la souris. La meilleure façon de faire est d&#39;utiliser un rasoir électrique, mais vous pouvez également utiliser des gels d&#39;épilation. Essuyez-les avec l&#39;alcool pour stériliserla peau après </li><li> Ensuite, dessinez votre suspension cellulaire dans une seringue. 23 à 27 aiguilles de calibre meilleur travail puisqu&#39;ils ne seront pas obstruer avec des cellules. Si l&#39;aiguille est trop importante (&lt;23 gauge) le tractus aiguille peut laisser un grand trou par lequel les cellules peuvent fuir arrière. </li><li> Injecter des cellules sous la peau dans le dos de la souris. Parce que la peau est assez lâche, utiliser votre pouce et l&#39;index pour pincer et étirer la zone que vous voulez injecter. Injectez sous la peau, en prenant soin de ne pas percer trop profondément. Aussi, essayez de garder l&#39;aiguille de glisser hors du site d&#39;injection tout en appuyant sur le piston: cela empêchera la création d&#39;un trou dans la peau par lequel les cellules peuvent fuir si vous essayez d&#39;injecter à nouveau. </li><li> Après les cellules sont injectés, attendre quelques heures afin de permettre la souris pour se réveiller et courir avant de re-anesthésier d&#39;imagerie par bioluminescence. Cela évite la toxicité isoflurane. </li></ol></li><li> Dans l&#39;imagerie par bioluminescence in vivo des cellules transplantées <ol><li> Pour l&#39;imagerie par bioluminescence des animaux entiers, nous ne l&#39;injection intrapéritonéale de D-luciférine à 375 mg / kg. Peser les animaux de calculer la dose avant l&#39;injection. </li><li> Injecter le D-luciférine dans le péritoine, juste à côté de la ligne médiane. Soyez prudent de ne pas aller trop profondément ou vous pouvez endommager les organes internes. Si la souris commence à se réveiller, le replacer dans la boîte de KO et attendez une minute ou deux. Pour les souris, la bioluminescence maximale survient généralement 15-40 minutes après l&#39;injection. </li><li> N&#39;oubliez pas de placer papier mat noir dans la boîte d&#39;imagerie pour aider à absorber toute la lumière n&#39;est pas émise par les CSEh. Placez la souris (jusqu&#39;à arrière) dans la chambre de l&#39;imagerie avec l&#39;isoflurane, sur le dessus du papier noir. Commencez avec un temps d&#39;exposition de 10 secondes. Si le signal est saturé, essayez de diminuer le temps d&#39;exposition; si elle est trop faible, augmenter l&#39;exposition. </li><li> Une fois le temps d&#39;exposition du signal a été optimisé pour votre souris, commencez à prendre des images à chaque minute jusqu&#39;à ce que le signal atteigne un maximum. Le mieux est fait en utilisant le logiciel d&#39;imagerie pour sélectionner &quot;des régions d&#39;intérêt (ROI)&quot; qui couvrent vos sites d&#39;injection. En surveillant l&#39;intensité du signal à chaque ROI, vous pouvez facilement déterminer si le signal commence à diminuer, ce qui indique que l&#39;intensité du signal maximal a été atteint. Le signal maximal devrait être utilisé comme vos données finales. </li><li> Lorsque vous êtes satisfait de vos images, enlever la souris de l&#39;isoflurane et de lui permettre de se réveiller dans sa cage. Habituellement, la souris doit se réveiller dans les 15 minutes. </li><li> L&#39;imagerie par bioluminescence gène rapporteur peut être répété quotidiennement, hebdomadaire et mensuelle pour aussi longtemps que les CSEh survivre dans l&#39;animal. Signal provenant d&#39;un tératome en développement augmentera de façon exponentielle au fil du temps, typiquement de l&#39;ordre de quelques semaines, comme le CSEh commencent à proliférer. </li><li> Après le cours du temps désiré d&#39;images de bioluminescence est acquise, l&#39;animal peut être sacrifié et des coupes de tissus utilisés pour l&#39;histologie. </li></ol></li></ol>

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Comparé à d&#39;autres modalités telles que la TEP et l&#39;IRM, la bioluminescence a limité la résolution spatiale et la pénétration tissulaire réduite en raison de l&#39;énergie relativement faible de photons émis (2-3 eV); pour ces raisons, il n&#39;a jusqu&#39;ici pas été applicables dans les grands animaux. Toutefois, la bioluminescence a l&#39;avantage d&#39;être à faible coût et à haut débit, et non invasive, ce qui rend hautement souhaitable pour les cellules souches in vivo de suivi dans les petits animaux. Gènes ...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<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>
</tr>
<tr>
<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6-well tissue culture-treated plates</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>92006</td>
<td>&nbsp;</td>		</tbody>
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in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

La découverte de cellules souches embryonnaires humaines (CSEh) a considérablement augmenté les outils disponibles pour les scientifiques médicaux s&#39;intéressent à la médecine régénérative. Cependant, l&#39;injection directe de CSEh, et les cellules différenciées de CSEh, dans les organismes vivants n&#39;a jusqu&#39;ici été entravée par la mort cellulaire importante, la formation de tératome, et le rejet immunitaire de l&#39;hôte. Comprendre le comportement en CSEh in vivo après transplantation nécessite des techniques d&#39;imagerie pour suivre longitudinalement la localisation de CSEh, la prolifération et la viabilité. L&#39;imagerie moléculaire a donné enquêteurs un moyen à haut débit, peu coûteux et sensibles pour le suivi de la prolifération cellulaire in vivo sur plusieurs jours, semaines, voire des mois. Ce progrès a considérablement augmenté la compréhension de la cinétique spatio-temporelle de la greffe de CSEh, la prolifération et tératome-formation chez des sujets vivants. Une avancée majeure dans l&#39;imagerie moléculaire a été l&#39;extension de la non-invasive des dosages gène rapporteur de la biologie moléculaire et cellulaire dans les plates-formes d&#39;imagerie in vivo la multi-modalité. Ces gènes rapporteurs, sous contrôle de promoteurs d&#39;ingénierie et exhausteurs qui tirent parti de la machinerie transcriptionnelle de la cellule hôte s, sont introduits dans les cellules en utilisant une variété de méthodes vectorielles et non vectorielles. Une fois dans la cellule, les gènes rapporteurs peuvent être transcrits soit de manière constitutive, ou seulement sous certaines conditions biologiques ou cellulaire, en fonction du type de promoteur utilisé. La transcription et la traduction des gènes rapporteurs dans les protéines bioactives est ensuite détecté grâce sensibles, l&#39;instrumentation non invasive (par exemple, caméras CCD) à l&#39;aide de signaux générant des sondes telles que D-luciférine. Afin d&#39;éviter le besoin de lumière excitatrice pour suivre in vivo les cellules souches est aussi nécessaire pour l&#39;imagerie de fluorescence, bioluminescence systèmes d&#39;imagerie du gène rapporteur exigent seulement une sonde exogène administrée pour induire une émission de lumière. Luciférase de luciole, dérivé de la pyralis firefly Photin, code pour une enzyme qui catalyse D-luciférine le métabolite actif optiquement, oxyluciférine. Activité optique peut alors être contrôlé avec une caméra CCD externes. Stablement transduites cellules qui transportent le journaliste de construire au sein de leur ADN chromosomique va passer le journaliste construction d&#39;ADN à des cellules filles, permettant un suivi longitudinal de la survie et la prolifération des CSEh in vivo. Par ailleurs, parce que l&#39;expression du produit du gène rapporteur est requis pour la génération du signal, seule solution viable cellules mère et fille, va créer le signal de bioluminescence; cellules apoptotiques ou morts ne sera pas. Dans cette vidéo, les matériaux spécifiques et les méthodes nécessaires pour la prolifération des cellules souches de suivi et de formation de tératome avec l&#39;imagerie par bioluminescence seront décrites.

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

Avec l&#39;intérêt croissant pour les thérapies de cellules souches, des techniques d&#39;imagerie moléculaire sont idéales pour le comportement des cellules souches de surveillance après la transplantation. Gènes rapporteurs luciférase ont permis non invasive, l&#39;évaluation répétitive de la survie cellulaire, l&#39;emplacement et la prolifération in vivo. Cette vidéo vous montrera comment suivre la prolifération des CSEh dans une souris vivante.

In vitro et in vivo en bioluminescence gène rapporteur imagerie des cellules souches embryonnaires humaines

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 vues.  Stanford University School of Medicine. Avec l'intérêt croissant pour les thérapies de cellules souches, des techniques d'imagerie moléculaire sont idéales pour le comportement des cellules souches de surveillance après la transplantation. Gènes rapporteurs luciférase ont permis non invasive, l'évaluation répétitive de la survie cellulaire, l'emplacement et la prolifération in vivo. Cette vidéo vous montrera comment suivre la prolifération des CSEh dans une souris vivante.

Vidéo: 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.

Dérivation des cellules souches embryonnaires humaines par 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 ...

Recherche

Biologie

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.

Palpage pour activité complexe mitochondrial dans les cellules souches embryonnaires humaines

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.

Marquage in vitro des cellules souches embryonnaires humaines pour l&#39;imagerie par résonance magnétique

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.

Culture et entretien des cellules souches embryonnaires humaines

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.

Congélation et de décongélation cellules souches embryonnaires humaines

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.

Vidéo analyse bioinformatique des droits de croissance de la tige colonie de cellules embryonnaires

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 différenciation des cellules souches embryonnaires en précurseurs d&#39;oligodendrocytes

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.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

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.

Reprogrammation des cellules somatiques humaines dans cellules souches pluripotentes induites (CISP) Utilisation vecteur rétroviral avec la GFP

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.

Isolement du ribosome Bound polypeptides naissants<em&gt; In vitro</em&gt; Pour identifier les sites de pause de translation le long d&#39;ARNm

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