Soutenu par: USPHS octroi NS38500, Institut national des troubles neurologiques et des maladies, NIH, USA; Alliance technologie biomédicale du sud-est du Wisconsin, Milwauke, WI, USA; Bryon Paralysie Riesch Fondation, Milwaukee, WI, USA; Ordre Fraternel des Aigles, Midwest chapitre , USA, National Association lésions médullaires, Milwaukee chapitre, USA; North East England Stem Cell Institute, Université de Newcastle, Royaume-Uni.

Dissection des Ardennes de follicules adultes Whisker souris <ol><li> Nous utilisons 10 semaines à 6 mois des souris âgées. Les jeunes souris produisent souvent plusieurs cellules. Euthanasier les animaux de la souris et plongez dans l&#39;ensemble du mélange 1:1 bétadine (solution d&#39;iode) et du peroxyde d&#39;hydrogène (disponible à la pharmacie) pendant environ 3 minutes. </li><li> Squirt souris entier, en particulier la région du visage, avec de l&#39;éthanol 75%, et de la souris pour effectuer microscope à dissection dans une hotte à flux laminaire. </li><li> Disséquer babines, en évitant soigneusement de couper en bulbes pileux, et piscine dans HBSS. </li><li> Disséquer follicules moustaches et une piscine dans HBSS à température ambiante. Pour ce faire, en tenant la peau à côté de follicule pileux avec une pince, puis découpe autour du follicule avec les ciseaux droits. Couper en profondeur pour éviter de blesser les bulbes pileux. </li><li> Ascenseur follicule moustaches de pad moustaches et mis en nouvelle plaque avec HBSS frais. </li><li> Rincer adipeux lâche et de tissu cutané avec jets de tampon, jusqu&#39;à ce que les follicules moustaches sont propres. Si nécessaire, couper nerf follicule moustaches et éliminer les tissus adhérentes par grattage et bouffées répétées de HBSS. </li><li> Pin follilce cheveux propres sur Sylgard verre enduit boîte de Petri en utilisant des aiguilles de tungstène affûté, en utilisant microforceps pour la tenue de la peau sur la partie à côté du follicule. </li><li> Moustaches coupées follicule longitudically avec un microlames. Évitez de couper trop profondément, car cela va blesser le bulbe. Retirer le sang gicle avec répétées de HBSS jusqu&#39;à allé. Apparition de sang est une bonne indication que la coupe était assez profonde. Si la coupe longitudial n&#39;est pas suffisamment longue, il peut être rallongé avec microciseaux plié. </li><li> Faites une coupe transversale au-dessus du niveau du sinus caverneux et ensuite une deuxième coupe transversale au niveau des sinus dans le ring, près de la peau. </li><li> Vous allez maintenant voir le renflement intérieur de la capsule. </li><li> Prenez une fin de capsule de collagène d&#39;avec la pince et le déploiement du bulbe avec une aiguille de tungstène plié. Vous allez maintenant voir la capsule vide et le renflement isolés. </li><li> Piscine isolés renflements dans une plaque de culture séparés dans HBSS à température ambiante. Assurez-vous qu&#39;aucun autre tissu contamine les renflements en commun. </li></ol> Culture d&#39;explants bulbe <ol><li> Manteau 35 mm des plaques de culture avec du collagène en plaçant 50 collagène ul et 10 ul stérile 6% de NaCl côté de l&#39;autre. Mélangez bien avec une pipette à double plié Pasteyr et pousser vers le bord de la plaque de culture. Incuber une nuit dans un dessiccateur propre, mais ne laissez pas sécher. Avant utilisation, rincer les plaques avec une solution saline. </li><li> Pré-incuber des plaques de culture revêtus de collagène et rincés pendant environ 3 heures avec un milieu de culture. Le milieu de culture est constitué de 85% d&#39;alpha-MEM modifiés, 10% de sérum de veau fœtal et 5% jour 11 d&#39;extrait d&#39;embryon de poulet. </li><li> Après ré-incubation, éliminer le milieu de culture à partir de plaques et d&#39;ajouter plusieurs renflements moyenne aussi peu que possible. Retirer l&#39;excès milieu de culture. </li><li> Incuber pendant 1 heure, mais pas plus, dans la cellule incubateur dans une atmosphère humidifiée avec de l&#39;oxygène 10% et 5% de CO 2. </li><li> Après 1 heure, doucement ajoutez 1,5 ml de milieu de culture. Explants Ardennes devraient adhérer au substrat de collagène. Remplacer 50% du milieu de culture quotidienne. </li><li> Dans les 3 - 4 jours, les cellules hautement migratoires vont émigrer de l&#39;explants renflement. Notez leur morphologie stellaire, la motilité, et l&#39;absence prédominante de contacts cellule-cellule. Au cours des prochains jours, plus de cellules d&#39;émigrer, et a émigré cellules vont proliférer rapidement. </li><li> Retirer renflement 2 - 3 jours après l&#39;apparition des EPI-NCSC l&#39;émigration avec une aiguille de tungstène affûté. Si les renflements sont laissés plus longtemps, ils ont tendance à s&#39;aplatir et à rendre impossible à obtenir purs EPI-NCSC cultures. </li><li> Important: rares cellules avec une morphologie aplatie, qui deviennent parfois apparente plusieurs jours plus tard EPI-NCSC, et qui sont moins mobiles ne sont PAS EPI-NCSC, mais putatifs cellules souches épidermiques / progéniteurs. Cultures composée de ces cellules, ou des cultures mixtes contenant du PEV-NCSC et putatifs cellules souches épidermiques / progéniteurs doivent être jetés. </li><li> Astuce: Ne pas garder les cellules pendant trop longtemps dans le milieu de explant primaire, car elles tendent à se différencier rapidement à haute densité cellulaire, en raison de putativement autocrine / paracrine de signalisation du facteur de croissance. </li></ol>

<ol>
	<li>Sieber-Blum, M., Grim, M., YF, H. u., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle.">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li><li>Sieber-Blum, M., Grim, M., Tuan, R., Grim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+hair+follicle+-+cradle+for+pluripotent+neural+crest+stem+cells.">The adult hair follicle - cradle for pluripotent neural crest stem cells.</a> Birth Defects Research (Part C). 72, 162-172 (2004).</li><li>Sieber-Blum, M., Schnell, L., Grim, M., Schneider, R., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+epidermal+neural+crest+stem+cell+(EPI-NCSC)+grafts+in+the+lesioned+spinal+cord.">Characterization of epidermal neural crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord.</a> Pub. 32, 67-81 (2006).</li><li>Hu, Y. F., Zhang, Z. -. J., Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epidermal+neural+crest+stem+cell+(EPI-NCSC)+molecular+signature.">An epidermal neural crest stem cell (EPI-NCSC) molecular signature.</a> Stem Cells. 24, 2692-2702 (2006).</li></ol>

En vertu de leur capacité migratoire, EPI-NCSC peut être isolé comme une population très pure de cellules souches, qui peuvent être développées in vitro. Comme vestiges embryonnaires dans un endroit adulte, EPI-NCSC sont des candidats potentiellement intéressants pour l&#39;avenir de thérapies de remplacement cellulaire, le génie biomédical et / ou la médecine régénérative. Nous avons testé le PEV-NCSC dans un modèle murin de lésion de la moelle épinière, où elles présentent des traits désirables. Grâce profilage de ...

mouse epidermal neural crest stem cell (epi-ncsc) cultures

EPI-NCSC sont des vestiges de la crête neurale embryonnaire dans un endroit adulte, le renflement des follicules pileux. Ce sont des cellules souches multipotentes qui ont la propriété physiologique de générer un large éventail de types de cellules différenciées, y compris les neurones, les cellules nerveuses de soutien, les cellules musculaires lisses, les os / les cellules du cartilage et des mélanocytes. EPI-NCSC sont facilement accessibles dans la peau velue et peut être isolé comme une population très pures de cellules souches. Cette vidéo fournit un protocole détaillé pour la préparation de la souris EPI-NCSC cultures de follicules moustaches. Le pad moustaches d&#39;une souris adulte est enlevée, et les follicules moustaches disséqués. Les follicules sont ensuite coupées longitudinalement et transversalement par la suite au-dessus et en dessous de la région de renflement. Le renflement est retiré de la capsule de collagène et placé dans une plaque de culture. EPI-NCSC commencent à émigrer de l&#39;explants renflement 3 à 4 jours plus tard.

Watch this Scientific Journal Video about Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures at JoVE.com

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Ici, nous montrons notre méthode pour isoler la souris épiderme des cellules souches neurales de crête (EPI-NCSC). Technique consiste à micro-dissection follicules moustaches, isoler le renflement et il placeing en culture de tissus. EPI-NCSC commencent à émigrer à partir d&#39;explants renflement sur le substrat dans les 3 - 4 jours.

Souris épidermique Neural Stem Cell Crest (EPI-NCSC) cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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Research

JoVE Journal

Biology

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

11.5K vues.  Newcastle University. Ici, nous montrons notre méthode pour isoler la souris épiderme des cellules souches neurales de crête (EPI-NCSC). Technique consiste à micro-dissection follicules moustaches, isoler le renflement et il placeing en culture de tissus. EPI-NCSC commencent à émigrer à partir d'explants renflement sur le substrat dans les 3 - 4 jours.

Vidéo: Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES Cell dérivé des cultures cellulaires neuroépithéliales

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Recherche

Biologie

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Culture de la souris de neurones précurseurs de cellules souches

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissection de 6,5 embryons de souris DPC

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Comptage des cellules souches humaines

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Évaluer la motilité Neural Stem Cell utilisant un gel d&#39;agarose à base dispositif microfluidique

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Génération d&#39;une seule cellule suspensions de tissus de souris de neurones

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Préparation du Pooled lysat plaquettaire humain (pHPL) comme un supplément efficace pour animaux sans sérum humain cultures de cellules souches

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolement et la dérivation de cellules germinales embryonnaires de souris

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Imagerie en temps réel de la motilité cellulaire et cytosquelette d&#39;actine des neurones individuels et de la crête neurale dans les embryons de poissons zèbres

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Suppression des fibroblastes rapide de haute densité embryonnaires humaines cultures de cellules souches

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...