Embryonnaires humaines études de cellules souches dans le laboratoire Teitell sont soutenus par un California Institute for Regenerative Medicine (CIRM) Subvention de démarrage RS1-00313. Nous remercions les membres du grand centre de médecine régénérative et de recherche sur les cellules souches à l&#39;UCLA, en particulier le Dr Clark Amander, le Dr Jerome Zack, et des membres de l&#39;UCLA large souches Facilité Institut noyau cellulaire pour leur soutien de nos études.

Fractionnement du Matrigel CSEh au Matrigel Habituellement, un confluentes 6-même assiette de CSEh sur Matrigel peut être divisé 1:03-1:05 à une autre plaque de Matrigel, les puits deviennent confluentes à nouveau 4-5 jours après fractionnement. <ol><li> CM et plaques Matrigel sont préparés comme décrit ci-dessus avant de se séparer. </li><li> Le jour de fractionnement, se laver chaque puits pour le fractionnement avec 1 x PBS, pH 7,4, ajoutez 1 ml de 1mg/ml dispase (une solution diluée d&#39;actions 5mg/ml avec DMEM/F12 médias) dans chaque puits et incuber à 37 ° C pendant 3 min. </li><li> Laver chaque puits trois fois avec 1 x PBS, pH 7,4, ajoutez 1 ml d&#39;ES médias sans bFGF, utiliser un grattoir à cellules de gratter les colonies du fond du puits, de recueillir les CSEh en suspension dans un tube de 50ml Falcon, et par centrifugation à 200g pendant 5 min à température ambiante. </li><li> Pour ré-plaque de l&#39;CSEh sur des plaques de Matrigel, d&#39;abord laver les assiettes avec Matrigel DMEM/F12 et ajouter 2,5 ml de CM complétée par 10ng/ml bFGF par puits. Reprendre le culot de CSEh dans un volume approprié de CM complété avec 10ng/ml bFGF, une pipette la suspension cellulaire de haut en bas trois fois. (REMARQUE: Lorsque les CSEh au fractionnement proviennent de plaques de Matrigel, les massifs sont très faciles à casser, donc trois séries de pipetage est assez et plus de pipetage peut perturber le cours-colonies et de faire des cellules individuelles.) Aliquote de 0,5 ml de suspension par des touffes de CSEh puits de la plaque de 6 puits Matrigel pour atteindre 3ml ainsi que par un volume final. Placer la plaque dans un incubateur à 37 ° C. </li></ol> Détection des marqueurs de pluripotence par immunofluorescence Immunofluorescence est utilisée pour examiner l&#39;expression des marqueurs de pluripotence des CSEh Oct-4, SSEA-4 en cours de culture. <ol><li> Aspirer les médias d&#39;un puits et laver avec 1 x PBS, pH 7,4. Ajouter 2 ml de formaldéhyde de 3,7% pour le bien de fixer pendant 10 min. </li><li> Rincer à l&#39;1xPBS, pH 7,4, ajouter 2 ml de méthanol pour le bien et le lieu à -20 ° C pendant 2 min. </li><li> Rincer à l&#39;1xPBS, pH 7,4, et bloquer avec 1ml de serume bovin à 0,2% d&#39;albumine (BSA) pendant 5 min. </li><li> Ajouter 1 ml d&#39;anticorps de Oct-4 ou anti-h/mSSEA-4 (1:200 dilution dans 0,2% de BSA), et incuber à température ambiante pendant 1 heure ou à 4 ° C pendant la nuit. (REMARQUE: pour SSEA-4 taches, passez directement à l&#39;étape 6) </li><li> Rincer trois fois avec 1xPBS, pH 7,4, ajoutez-FITC conjugué IgG de lapin (1:500 dilution dans 0,2% de BSA), et incuber à température ambiante pendant 1 heure. </li><li> Rincer 3 fois avec 1xPBS, pH 7,4, et de mettre une goutte de solution de montage (peut contenir DAPI, si la visualisation nucléaire est souhaitée) au centre du puits. Mettez la lamelle sur le dessus des cellules, sceller avec du vernis à ongles, et d&#39;évaluer par microscopie d&#39;immunofluorescence. </li></ol> Embryonnaires humaines reçus de cellules souches ES médias (cellules souches embryonnaires): DMEM/F12 - 400ml Knockout Sérum Replacer - 100ml Non-acides aminés essentiels - 5ml 200 mM glutamax / BME Solution - 5ml Pénicilline / streptomycine - 5ml Mélangez bien et stocker finale Médias Culture CSEh dans des bouteilles de 500ml à 4 ° C. Bon pour un maximum de deux semaines. Culture MEF médias: DMEM - 1000ml FBS - 100ml Non-acides aminés essentiels - 10ml Glutamine - 10ml Penicllin / Streptomycine - 10ml Mélangez bien et stocker finale Médias Culture MEF à 4 ° C. 200 mM Glutamax / BME Solution: Glutamax - 100ml β-mercaptoéthanol (solution stock à 14,3) - 140 μ1 Mélangez deux solutions bien et puis des portions de 10ml aliquote. Gardez aliquotes à -20 ° C. bFGF solution (concentration finale de stock: 10 microgrammes / ml): bFGF (FGF2) - 50 mg 0,1% de BSA dans les 1xPBS, pH 7,4 - 5 ml Dissoudre 50 microgrammes de bFGF dans 0,1% de BSA 5ml de faire un 10 ug / ml stock. Aliquoter en portions de 500 ul. Conserver à -80 ° C. Collagénase IV Solution (1mg/ml): Collagénase IV - 50mg DMEM/F12 Médias - 50ml Ajouter la poudre de collagénase IV à un tube de 50ml Falcon. Dans la hotte, mélanger dans 50ml de stériles DMEM/F12 médias. Vortex la solution pour &lt;1 min. pour s&#39;assurer que la poudre soit dissoute. Dans la hotte, passer par un filtre de 0,22 um dans un nouveau tube 50ml stérile faucon. Cette solution est bonne pour un maximum de 2 semaines à 4 ° C. Dispase Solution (1mg/ml): Dispase 5mg/ml - 2ml Médias DMEM/F12 - 8ml Aliquoter solution commerciale Dispase 5mg/ml et conserver à -20 ° C. Diluer dispase à 1mg/ml utilisant DMEM/F12. Cette solution peut être conservée pendant 1 semaine à 4 ° C.

<ol>
	<li>Thomson, J. a. m. e. s. A., Itskovitz-Eldor, J. o. s. e. p. h., Shapiro, S. a. n. d. e. r. S., Waknitz, M. i. c. h. e. l. l. e. A., Swiergiel, J. e. n. n. i. f. e. r. J., Marshall, V. i. v. i. e. n. n. e. S., Jones, J. e. f. f. r. e. y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts+.">Embryonic Stem Cell Lines Derived from Human Blastocysts .</a> Science. 282, 1145-1147 (1998).</li><li>Xu, C. h. u. n. h. u. i., Inokuma, M. a. r. g. a. r. e. t. S., Denham, J. e. r. r. o. d., Golds, K. a. t. h. a. l. e. e. n., Kundu, P. r. a. t. i. m. a., Gold, J. o. s. e. p. h. D., Carpenter, M. e. l. i. s. s. a. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells+Nature+Biotechnology+19."> Feeder-free growth of undifferentiated human embryonic stem cells Nature Biotechnology 19.</a> Nature Biotechnology. 19, 971-974 (2001).</li></ol>

Cette série de 3 vidéos démontre comment faire pousser des cellules souches embryonnaires humaines (CSEh) sur fibroblastes de souris embryonnaires des cellules nourricières (MEF) (vidéo 1), comment les passage à Matrigel sans cellule d&#39;alimentation des plaques (vidéo 2), et comment maintenir CSEh par passage dans le chargeur Matrigel sans cellules conditions (vidéo 3). De nombreuses études antérieures ont montré que l&#39;entretien de viable, CSEh indifférenciées nécessite la culture sur cellules nourricières inactivées MEF. Cependant, pour de nombreuses expériences, une population pure de CSEh libre de toute contamination de cellules nourricières est nécessaire. Pour atteindre cet objectif, nous avons démontré comment les CSEh passage de plaques d&#39;alimentation au chargeur de pile MEF plaques sans Matrigel et la façon de maintenir et de façon continue des colonies de CSEh passage dans le chargeur conditions acellulaires. En suivant ce protocole, une petite quantité de contamination de cellules nourricières MEF pourrait encore exister au passage de l&#39;un sur une plaque de Matrigel, mais ce contaminant est généralement facile à visualiser. Au passage de Matrigel deux et au-delà, aucune contamination du MEF se trouve généralement, laissant une population pure de CSEh pour des expériences. Immunofluorescence et cytométrie de flux pour microscopie ou marqueurs de pluripotence des CSEh, tels que Oct-4, SSEA-4, sont nécessaires pour confirmer l&#39;entretien des CSEh dans un état indifférencié pendant la culture.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Knockout Serum Replacer (KSR)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td> Non-essential Amino Acids</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMax </td>
<td>Reagent</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11995-065</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Clontech</td>
<td>631107</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030-081</td>
<td>&nbsp;</td>
<tr>
<td>BME</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP176-100</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>233-FB-025</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>Reagent</td>
<td>Gibco</td>
<td>17104-019</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>Reagent</td>
<td>Stem Cell Technologies</td>
<td>17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin</td>
<td>Reagent</td>
<td>Chemicon</td>
<td>ES-006-B</td>
<td>&nbsp;</td>
<tr>
<td>Matrigel</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>354277</td>
<td>&nbsp;</td>
<tr>
<td>Oct-4 antibody</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>SC-9081</td>
<td>&nbsp;</td>
<tr>
<td>anti-h/mSSEA-4 Phycoerythrin Conjugated Mouse IgG3</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>FAB1435P</td>
<td>&nbsp;</td>
<tr>
<td>FITCI-conjugated antirabbit IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch Laboratories. Inc.</td>
<td>715-095-152</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>

from mefs to matrigel 3: passaging hescs from matrigel onto matrigel

Cette vidéo montre comment maintenir la croissance de cellules souches embryonnaires humaines (CSEh) dans le chargeur sans cellules conditions et la manière de CSEh passage en continu dans le chargeur sans cellules conditions. Confirmation de CSEh pluripotence grandi dans le chargeur sans cellules par microscopie d&#39;immunofluorescence conditions est également démontrée.

Watch this Scientific Journal Video about From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel at JoVE.com

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

De FAE au Matrigel 3: Le repiquage du Matrigel CSEh sur Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

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13.7K vues.  University of California, Los Angeles. Cette vidéo montre comment maintenir la croissance de cellules souches embryonnaires humaines (CSEh) dans le chargeur sans cellules conditions et la manière de CSEh passage en continu dans le chargeur sans cellules conditions. Confirmation de CSEh pluripotence grandi dans le chargeur sans cellules par microscopie d'immunofluorescence conditions est également démontrée.

Vidéo: From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

HUES repiquage souches embryonnaires humaines lignées cellulaires à la trypsine

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

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Biologie

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Le repiquage des cellules souches humaines

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

De MEFs à I Matrigel: CSEh repiquage en présence du MEF

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Générer des cellules iPS de MEFS travers l&#39;expression forcée de Sox-2, Oct-4, c-Myc et Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

De FAE au Matrigel 2: Fractionnement des CSEh MEFs sur Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Chargement du fret sur la kinésine Powered navettes moléculaires

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Une introduction au laboratoire Worm: La culture de Worms à la mutagenèse

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Un test basé sur Matrigel Tube Formation d&#39;évaluer l&#39;activité Vasculogenic de cellules tumorales

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

La production efficace et purification de recombinant murin Kindlin-3 à partir de cellules d&#39;insectes pour les études biophysiques

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Génération de Myospheres De CSEh par Reprogrammation épigénétique

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

De FAE au Matrigel 3: Le repiquage du Matrigel CSEh sur Matrigel

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