Les auteurs remercient Janet Rossant pour le protocole initial. Cette étude a été soutenue par l&#39;Institut national de la Santé accorde CA106308 à WH.

Dans ce protocole, nous décrivons la préparation de blastocystes de souris et de la création de lignées de cellules TS. Manipulations de souris générale préalable à la collecte de blastocystes, y compris la mise en place pour l&#39;accouplement naturel et l&#39;induction de l&#39;ovulation super, essentiellement suivi le protocole standard illustré par Nagy et al. 2003 (pp146-150). Dérivation et la maintenance des cellules TS <ol><li> Croisées entre les souris d&#39;élevage de configuration d&#39;intérêt. </li><li> Préparer les fibroblastes embryonnaires de souris (MEF) en tant que cellules nourricières. Deux jours avant la collecte des blastocystes, FAE (2 x 10 6 cellules) sont étalées dans des boîtes de 100 mm. Le lendemain, ces cellules sont traitées avec 10 ml de milieu de TS contenant la mitomycine C (20 mg / ml) pendant 2 heures d&#39;incubation à 37 ° C, 5% de CO 2. Il est suivi par un lavage des cellules deux fois avec du PBS. Les cellules mitomycine C traitées sont ensuite ensemencées dans des plaques 12 puits (1 x 10 5 cellules / puits). </li><li> Le jour de la collecte de blastocyste (3,5 jpc, appelé le premier jour), un milieu de culture est remplacé par le MEF TS moyenne plus de 1x FGF4/Heparin (0,5 ml / puits). </li><li> Préparation pour la collecte de l&#39;utérus: euthanasier la souris et de localiser l&#39;utérus (figure 1A). </li><li> Enlever l&#39;utérus en la saisissant avec une pince à col de l&#39;utérus (situé derrière la vessie) et couper à travers la jonction de l&#39;utérus et du col utérin (figure 1B). Tirez l&#39;utérus, les garnitures de la membrane et les tissus adipeux loin et couper l&#39;utérus en dessous de la jonction avec l&#39;oviducte (figure 1C). Placez le col (figure 1D) dans un petit volume (0,2 ml) du milieu TS dans une plaque de 60 mm. </li><li> Rincer chaque corne utérine avec 1 ml de milieu de TS à l&#39;aide d&#39;une seringue de 1 ml avec une aiguille de calibre 26. Insérez l&#39;aiguille dans la base de l&#39;utérus et rincer lentement dans une nouvelle plaque de 60 mm avec une moyenne TS ml. L&#39;utérus doit être gonflé et étendu intégralement pendant le rinçage. </li><li> Recueillir et le transfert des blastocystes avec précaution dans un endroit propre plaque 12 puits contenant du milieu TS à l&#39;aide d&#39;une pipette bouche-contrôlé. Lavez blastocystes à trois reprises par le transfert de série par le biais du milieu TS en plaques de 12 puits. Placez un blastocyste par puits (figure 2A) dans une plaque de 12 puits contenant les mitomycine C-traitée mangeoires MEF et la culture à 37 ° C, 5% de CO 2. </li><li> Les blastocystes éclosent à partir zone pellucide et joindre les puits de 24 à 36 heures. Une petite excroissance être facilement observée le jour 3. Nourrissez les cultures avec 500 ul du milieu TS frais plus 1x FGF4/Heparin. </li><li> Au jour 4 ou 5, l&#39;excroissance blastocyste devrait être prêt pour la ventilation en fonction de sa taille. La taille idéale pour la désagrégation des cellules TS est illustré dans la figure 3B. Laver les cultures une fois avec 0,5 ml de PBS. Ajouter 100 ml 0,25% de trypsine / EDTA et incuber pendant 5 minutes à 37 ° C, 5% de CO 2. Pause dans l&#39;excroissance de petites touffes d&#39;aspiration et refoulement vigoureusement avec une pipetteman P100. Arrêter le traitement à la trypsine en ajoutant le TS-70CM-1.5F4H (milieu TS 30%, 70% MEF-CM, plus FGF4/Heparin 1.5x) dans le puits. Changer moyenne 8 heures après la désagrégation et de continuer à nourrir les cellules tous les 2 jours. </li><li> Entre les jours 6 et 10 (très variable) des colonies de cellules TS peuvent être observés. Ils ont plat, drap, comme la morphologie épithéliale avec une frontière claire colonie (figure 2C). Continuer à alimenter les cultures tous les 2 jours </li><li> Lorsque les cellules atteignent la confluence TS de 50% (généralement entre 15 et 20 jours), se laver une fois avec du PBS et ajouter 100 ml 0,25% de trypsine / EDTA. Pipeter haut en bas durant trypsinisation pour assurer une proximité unique de cellules en suspension. Arrêter le traitement à la trypsine en ajoutant le TS-70CM-1.5F4H et le transfert de nouvelles plaques de 6 puits ou 60mm contenant la mitomycine C-traitée mangeoires MEF (2,5 x 10 5 cells/6-well ou 5 x10 5 cells/60mm plaque). </li><li> Changer moyenne 8 heures après le passage et continuer à nourrir les cultures tous les 2 jours. </li><li> Après un ou deux passages plus (3-5 jours entre les passages) sur les départs du MEF, les cellules peuvent être cultivées TS sans eux. Parce MEFs attacher à la plaque de culture beaucoup plus vite que les cellules TS après trypsinisation, une telle différence dans le taux adhérentes peuvent être utilisées pour séparer les cellules TS à partir des mangeoires MEF. Après le passage des cellules cultivées, les incuber à 37 ° C CO, 5% 2 pendant 30 min. La plupart des FAE se joindre à la plaque et la population TS reste en suspension. Transférer le surnageant dans une nouvelle plaque. Répétez ce processus pour plusieurs passages pour obtenir une population TS pur. </li><li> Maintenir les cellules TS dans le TS-70CM-1.5F4H et les passer tous les 4 jours (1:10-1:20 split) ou lorsque la culture atteint 70% de confluence. Passant les cellules à haute densité (supérieure à diviser 01h05) ou en les cultivant en trop confluentes peut provoquer leur différenciation. </li><li> L&#39;identité des cellules TS peut être confirmée par immunomarquage d&#39;un marqueur de cellule, Cdx2 (figure 3). </li></ol> TS cellules de congélation <ol><li> Préparer 2x milieu de congélation (50% de FBS, 30 moyennes TS% et 20% de DMSO). </li><li> Cocellules TS llect par trypsinisation, suivie par une centrifugation à 450g pendant 6 min. Les remettre en suspension dans un milieu de TS avec un volume égal de milieu de congélation 2x. </li><li> Congelez les cellules lentement en utilisant une chambre de l&#39;alcool isopropylique à -70 ° C et transfert à l&#39;azote liquide après 24-48 heures. </li><li> Une plaque de 60 mm (70% de confluence) est habituellement conservée dans deux flacons. </li></ol> La décongélation des cellules TS <ol><li> Vite dégeler le flacon à 37 ° C, les recueillir dans 10 médias TS ml par centrifugation (450g, 6 min), et remettre en suspension dans 30% TS médias, 70CM-1.5F4H. </li><li> Un flacon de cellules est ensemencé dans une plaque de 60 mm. </li></ol> Différenciation des cellules TS en TGC La différenciation des cellules TS en cellules géantes du trophoblaste (PGT) peut être induite par le retrait du MEF-CM, FGF4 et l&#39;héparine. Le phénotype TGC (gros noyaux) peut être détecté quatre jours après l&#39;induction (Tanaka et al., 1998 et Chiu et al. 2008). Immunocoloration des P450scc détecter l&#39;expression de ce marqueur TGC dans la culture différenciés (figure 4A). Cytométrie de flux IP (iodure de propidium) cellules colorées montre en outre la présence de cellules différenciées contenant plus forte teneur en ADN (figure 4B). Ces résultats suggèrent que les cellules subissent TS endoréduplication devenir TGC polyploïdes. Les résultats représentatifs <img src="/files/ftp_upload/1964/1964fig1.jpg" alt="Figure 1" /> Figure 1. Dissection de l&#39;utérus de la souris. (A) Situation de l&#39;utérus est indiqué par des flèches. (B) Couper à travers la jonction de l&#39;utérus et le col comme indiqué. (C) Couper l&#39;utérus juste en dessous de l&#39;oviducte (DO) au-dessous de l&#39;ovaire (VO). (D) Image de l&#39;utérus. <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="Figure 2" /> Images représentant la figure 2. Illustrent blastocyste de souris, excroissance blastocyste et la colonie du trophoblaste. (A) un blastocyste de souris recueillies à E3.5 est montré. ICM, la masse cellulaire interne, MT, trophectoderme mural; PT, trophectoderme polaire. (B) une excroissance du trophoblaste sur les départs du MEF est montré. TS, cellules souches trophoblastiques. (C) Une colonie de cellules souches trophoblastiques montre un net avantage sur les départs du MEF. Les flèches indiquent les limites des cellules. <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="Figure 3" /> Figure 3. Identification des cellules TS par immunomarquage analyse. TS (AD) et ES (contrôle, EH), les cellules sont soumises à l&#39;identification des marqueurs. Les cellules sont immunocolorés avec des anticorps de reconnaître Oct4 (A, E) ou Cdx2 (B, F), et contre par DAPI (C, G) a présenté en images fusionnées (D, H). La barre d&#39;échelle, 50 um. <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="Figure 4" /> Figure 4. Différenciation des cellules TS in vitro. (A) indifférenciées (Undiff) ou différenciés (Diff) de cellules en culture ont été examinés pour l&#39;expression d&#39;un marqueur de TGC, P450scc (vert), de contraste avec DAPI (en rouge). (B) Analyse par cytométrie en flux des cellules différenciées, tachée de PI, le contenu des mesures ADN (M1, deux à quatre exemplaires, M2, plus de quatre exemplaires). La population M2 représente les cellules trophoblastiques polyploïdes.

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

Dans cette vidéo, nous démontrons le processus de collecte E3.5 blastocystes à partir des procédures de l&#39;utérus et d&#39;expérimentation pour établir des lignées de cellules TS. Nous décrivons également la condition de maintenir le caractère souche de cellules TS et d&#39;induire leur différenciation en cellules différenciées. Deux étapes essentielles pures pour obtenir des lignées de cellules TS sont le moment de la désagrégation prolongement (étape 9) et le traitement du premier passage (étape...

Réactifs Milieu TS: RPMI-1640, 20% de FBS, 1 mM pyruvate de sodium, 100 mM de bêta-mercaptoéthanol et 100 mg / ml de pénicilline-streptomycine. Mitomycine C: Dissoudre 2 mg de mitomycine C (Sigma M-0503) dans 2 ml de PBS et ajouter ce mélange de 2 ml dans 200 ml TS moyenne (10 ng / ml). Faire 20 aliquotes (10 ml) et conserver à -20 ° C jusqu&#39;à utilisation. Ajouter une aliquote par 100 mm Plaque. Fibroblastes embryonnaires de souris-état moyen (MEF-CM): Plaque de MEFs plaques de 100 mm (1 x 10 6 / plaque). Le lendemain, ajoutez 10 ml TS moyenne par plat et continuer à la culture pendant 48 heures. Recueillir le milieu de culture, de les filtrer (0,45 mm) et conserver à -20 ° C dans 35 ml aliquotes. Dégel chaque aliquote lorsque cela est nécessaire, qui peut être conservé à 4 ° C. La FAE peut être utilisé pour préparer plus de deux lots de CM avant qu&#39;ils ne deviennent confluentes. PBS / BSA: Dissoudre BSA fraction, V (Sigma A3311, 0,1% (p / v)) dans du PBS (10 ml), filtrer à travers un filtre de 0,45 mm seringue et faire des aliquotes de 1 ml dans des tubes de 1,5 ml. Stocker à -70 ° C et le dégel d&#39;un tube en cas de besoin pour préparer FGF4. FGF4 (1000x): Resuspendre lyophilisé FGF4 (Sigma F8424, 25 mg) avec 1 ml de la solution de PBS / BSA. Mélangez bien et faites des aliquots de 10 (100 ml) dans des tubes de 1,5 ml et conserver à -70 ° C. Dégel chaque tube en cas de besoin et de stocker le reste à 4 ° C, ne pas recongeler. Diluer 1000 fois dans du milieu TS pour obtenir 1x FGF4 (25 ng / ml). L&#39;héparine (1000x): Resuspendre héparine (Sigma H3149, 10.000 unités) dans du PBS à une concentration finale de 1 mg / ml (1000x). Faire des aliquots de 100 ml dans des tubes de 1,5 ml et conserver à -70 ° C. Aliquotes dégel si nécessaire et stocker le reste à 4 ° C, ne pas recongeler. Diluer 1000 fois dans le milieu de TS pour obtenir 1x héparine (1 ng / ml).

derivation of mouse trophoblast stem cells from blastocysts

Spécification du trophectoderme est l&#39;un des événements les plus anciens de différenciation du développement des mammifères. La lignée dérivée de trophoblaste trophectoderme médie l&#39;implantation et génère la partie foetale du placenta. En conséquence, le développement de cette lignée est essentielle pour la survie des embryons. Dérivation de la tige du trophoblaste (TS), les cellules à partir de blastocystes de souris a été décrite par Tanaka<em&gt; Et al.</em&gt; 1998. La capacité des cellules TS afin de préserver la propriété trophoblaste spécifiques et leur expression de la scène et du type de cellules de marqueurs spécifiques après stimulation adéquate fournit un système modèle intéressant d&#39;étudier le développement lignée trophoblaste lequel récapitulant les événements placentation tôt. Par ailleurs, les cellules trophoblastiques sont l&#39;un des rares types de cellules somatiques en cours d&#39;amplification du génome naturel. Bien que les voies moléculaires qui sous-tendent polyploïdisation du trophoblaste ont commencé à se dénouer, le rôle physiologique et l&#39;avantage de l&#39;amplification du génome du trophoblaste reste largement insaisissable. Le développement des cellules souches diploïdes dans les cellules trophoblastiques polyploïdes dans la culture fait de ce<em&gt; Ex vivo</emSystème&gt; un excellent outil pour élucider le mécanisme de régulation de la réplication du génome et de l&#39;instabilité dans la santé et la maladie. Nous décrivons ici un protocole basé sur les rapports antérieurs à la modification publiée dans Chiu<em&gt; Et al.</em&gt; 2008.

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Dans cette vidéo, nous démontrons l&#39;isolement des blastocystes de souris et de la dérivation de cellules souches trophoblastiques à partir de blastocystes. Nous décrivons également les conditions d&#39;entretien de la propriété de cellules souches ainsi que l&#39;induction de la différenciation en culture.

Dérivation de cellules souches de la souris Trophoblast à partir de blastocystes

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

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18.1K vues.  University of Rochester. Dans cette vidéo, nous démontrons l'isolement des blastocystes de souris et de la dérivation de cellules souches trophoblastiques à partir de blastocystes. Nous décrivons également les conditions d'entretien de la propriété de cellules souches ainsi que l'induction de la différenciation en culture.

Vidéo: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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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 ...

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 ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

L&#39;isolement des cellules souches rétiniennes de l&#39;Oeil de la souris

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Isolement des cellules trophoblastiques primaires de souris et le test invasion trophoblastique

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Génération de souris dérivées de cellules souches pluripotentes induites

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Enrichissement de série de souches et des cellules progénitrices des spermatogonies (SSC) dans la culture de dérivation de long terme Lines adultes CSE de souris

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolement et culture des cellules souches épithéliales dentaire de l&#39;adulte de souris Incisive

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...