1. Préparation Régent <ol><li> Tampon: 1% (p / v) de sérum albumine bovine dans une solution saline équilibrée de Hank </li><li> Reconstituer les enzymes. L&#39;enzyme hyaluronidase: 40mg / ml, dissous dans du tampon. Collagénase II: 23 mg / mL, dissous dans du tampon. Utiliser des solutions fraîchement préparées enzyme fraîche pour chaque isolement. Une fois dissous, conserver à 4 ° C jusqu&#39;à leur utilisation pour la digestion. </li><li> 50 mM de chlorure de calcium dans l&#39;eau distillée. Stériliser par filtration à travers un filtre de 0,2 uM taille des pores. </li><li> Souris moyennes salivaires des glandes de la culture (MSG): DMEM: F12 avec de la pénicilline (100 UI / ml), streptomycine (100 ug / ml), glutamax (2 mM), facteur de croissance épidermique-2 (20 ng / ml), facteur de croissance des fibroblastes l&#39;insuline -2 (20 ng / ml), N2 complément (1%), (10 pg / ml) et la dexaméthasone (1 uM). </li></ol> 2. Digestion des tissus mécanique et enzymatique <ol><li> Peser les glandes salivaires disséquées. </li><li> Hacher les glandes en une pâte homogène à l&#39;aide des ciseaux de dissection stériles courbes dans une petite boîte de Petri. </li><li> Recueillir tissus hachés dans 14 ml tubes, en utilisant 1 ml de tampon par les tissus sous-maxillaire 80mg. Rincez les boîtes de Pétri nettoyage de tissus en utilisant certains des tampons. </li><li> Ajouter un autre 1 mL de tampon par 80 mg de tissu, suivis de 25 uL solution de collagénase enzyme II, 25 uL solution enzyme hyaluronidase et 250 uL solution de chlorure de calcium par 80 mg de tissu. Si vous travaillez avec de grandes quantités de tissu, les étapes 2.4 à 2.9 peuvent être effectuées dans des flacons T25 culture de tissu pour plus de commodité. </li><li> Incuber dans un bain-marie secouant fixée à 37 ° C pendant 20 minutes. Retirer les tubes et les triturent à la pipette pour mélanger soigneusement l&#39;enzyme à travers le tissu nouveau. </li><li> Remplacer dans un autre bain-marie pendant 20 min. </li><li> Recueillir les tissus par centrifugation à 400 xg, pendant 8 minutes. Rejeter le surnageant. </li><li> Remettre en suspension dans 2 mL de tampon pour chaque tissu de 80 mg, et répétez l&#39;enzyme et l&#39;ajout de chlorure de calcium comme ci-dessus. Incuber 20 minutes en secouant bain-marie. Retirer les tubes et les triturent à la pipette pour mélanger soigneusement les enzymes. </li><li> Incuber pour la finale de 20 min en secouant bain-marie. Recueillir les cellules par centrifugation comme ci-dessus, éliminer le surnageant. </li></ol> 3. Étapes de lavage <ol><li> Resuspendre chaque 80 mg de tissu en 2 mL de tampon et une pipette de laver le tissu sans enzymes. </li><li> Centrifuger comme précédemment à collecter. Rejeter le surnageant. </li><li> Répétez les laver en utilisant une mémoire tampon mL par 80 mg de tissu. Centrifugeuse à recueillir, éliminer le surnageant. </li></ol> 4. Filtrage <ol><li> Solution de tissus Remettre en suspension dans 1 ml de tampon par 80 mg de tissu. </li><li> Ajouter la solution à 100 um taille des pores du filtre placé sur le tube Falcon de 50 ml. Ne pas appliquer plus de 3 ml de solution tissus hachés par colonne, comme des filtres peuvent être bloquées. Laisser s&#39;infiltrer à travers. Enlever la matière filtrée accroché sur le dessous du filtre à la pipette, et ajouter à filtrat. </li><li> Utilisez une seringue avec une aiguille de calibre 26 pour prendre filtrat de 50 ml tubes et d&#39;appliquer des filtres à 50 um taille des pores sur les tubes de 5 ml. Laisser filtrer à travers, en aidant en desserrant les couvercles si nécessaire. </li><li> Tubes à centrifuger comme précédemment à collecter. Rejeter le surnageant. </li></ol> 5. Placage et moyennes <ol><li> Combiner tous les pastilles dans un seul volume. Comptez utilisant compteur de cellules automatisé ou hémocytomètre. </li><li> Cellules de la plaque à une densité de 0,4 x 10 6 cellules par puits de 12 puits plaque, ou 2,67 x 10 6 cellules par flacon de culture tissulaire T25. Ajouter une moyenne MSG ml à chaque puits ou 6 ml dans chaque flacon T25. </li><li> Incuber à 37 ° C. Sphères doit être clairement visible par jour 2. </li></ol> 6. Les résultats représentatifs: Après deux à trois jours de culture, de petits agrégats de cellules (salispheres) sera apparente dans les cultures. Salispheres va continuer à croître en taille, sur une période de dix jours dans la culture. Représentant des images de microscopie à contraste de phase d&#39;salispheres sont présentés dans la figure 1. Les cellules en prolifération exprimant des protéines marqueurs de cellules souches associées peuvent être isolés à partir de ces sphères, de manière optimale entre l&#39;isolement jours après 3-5, et sont capables de différenciation en cellules fonctionnelles salive, acineuses produisant. <img src="/files/ftp_upload/2484/2484fig1.jpg" alt="Figure 1" /> Figure 1. Salisphere la formation in vitro. Après la digestion mécanique et enzymatique en utilisant le protocole actuel, les sphères de la taille croissante peut être trouvé dans les cultures flottant. Les panneaux sont représentatives d&#39;images de microscopie à contraste de phase de sphères de quelques jours 0 (A), 4 (B), 7 (C) et 10 (D). Barre d&#39;échelle = 50 microns.

<ol>
	<li>Vissink, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Sequelae+of+Head+and+Neck+Radiotherapy.">Oral Sequelae of Head and Neck Radiotherapy.</a> Crit Rev Oral Biol Med. 14, 199-212 (2003).</li><li>Nape&amp;ntilde, a. s., J, J., Brennan, M. T., Fox, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+xerostomia+(dry+mouth).">Diagnosis and treatment of xerostomia (dry mouth).</a> Odontology. 97, 76-83 (2009).</li><li>Denny, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenchymal+cell+proliferation+and+mechanisms+for+maintenance+of+granular+duct+and+acinar+cell+populations+in+adult+male+mouse+submandibular+gland.">Parenchymal cell proliferation and mechanisms for maintenance of granular duct and acinar cell populations in adult male mouse submandibular gland.</a> 235, 475-485 (2005).</li><li>Man, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+a+perinatal+cellular+phenotype+in+submandibular+glands+of+adult+rat.">Persistence of a perinatal cellular phenotype in submandibular glands of adult rat.</a> J. Histochem. Cytochem. 43, 1203-1215 (1995).</li><li>Cotroneo, E., Proctor, G. B., Carpenter, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+acinar+cells+following+ligation+of+rat+submandibular+gland+retraces+the+embryonic-perinatal+pathway+of+cytodifferentiation.">Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation.</a> Differentiation. 79, 120-130 (2010).</li><li>Eirew, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+quantifying+normal+human+mammary+epithelial+stem+cells+with+in+vivo+regenerative+ability.">A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability.</a> Nat Med. 14, 1384-1389 (2008).</li><li>Gorjup, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glandular+tissue+from+human+pancreas+and+salivary+gland+yields+similar+stem+cell+populations.">Glandular tissue from human pancreas and salivary gland yields similar stem cell populations.</a> European Journal of Cell Biology. 88, 409-421 (2009).</li><li>Alonso, L., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+of+the+skin+epithelium.">Stem cells of the skin epithelium.</a> Proc Natl Acad Sci U S A. 100, 11830-11835 (2003).</li><li>Lombaert, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+salivary+gland+function+after+stem+cell+transplantation+in+irradiated+glands.">Rescue of salivary gland function after stem cell transplantation in irradiated glands.</a> Plos One. 3, e2063-e2063 (2008).</li><li>Coppes, R. P., Goot, A. v. a. n. d. e. r., Lombaert, I. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Therapy+to+Reduce+Radiation-Induced+Normal+Tissue+Damage.">Stem Cell Therapy to Reduce Radiation-Induced Normal Tissue Damage.</a> Seminars in Radiation Oncology. 19, 112-121 (2009).</li></ol>

La méthode de culture de tissus décrit ici représente un protocole reproductible pour l&#39;isolement des cellules souches contenant salispheres des glandes salivaires de souris. Les études utilisant des cellules isolées de cette manière ont mis en évidence la capacité de régénération de la glande salivaire cellules souches 9. La transplantation d&#39;une centaine de c-kit + cellules dérivées de la salispheres induite récupération fonctionnelle des glandes salivaires de souris irradiées. Ces données sont intéressantes et fournissent un point de départ pour l&#39;enquête sur les cellules souches pour la thérapie basée xérostomie. Beaucoup de pistes restent à explorer cependant, y compris le profil d&#39;expression marqueur protéique complète des cellules souches, la capacité des glandes sous-maxillaires pour sauver la fonction des glandes parotides irradiés salivaires et vice versa, et la caractérisation de la putatifs dans une niche de cellules souches in vivo des les cellules. Finalement, la traduction de ce protocole d&#39;échantillons de tissus humains et le potentiel ultérieur pour le traitement de la xérostomie chez les patients humains en utilisant des cellules isolées est l&#39;application la plus excitante de la méthode décrite.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3506</td>
<td>Store at &#x2013; 20 &deg;C</td>
</tr>
<tr>
<td>Collagenase II</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17101-015</td>
<td>Store at 4 &deg;C</td>
</tr>
<tr>
<td>Epidermal Growth Factor-2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E9644</td>
<td>Make a stock of 10 &mu;g / mL in phosphate buffered saline (PBS). Store at &#x2013; 20 &deg;C in single use aliquots.</td>
</tr>
<tr>
<td>Fibroblast Growth Factor-2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>Make stock of 25 &mu;g / mL in PBS. Store at &#x2013; 20 &deg;C in single use aliquots.</td>
</tr>
<tr>
<td>N2 supplement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17502-048</td>
<td>As manufacturer instructions.</td>
</tr>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I6634</td>
<td>Make stock of 2 mg / mL in tap water. Adjust water to pH 2-3 using glacial acetic acid prior to dissolving.</td>
</tr>
<tr>
<td>Dexamethasone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4902</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;M pore-size sterile cell strainers</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352360</td>
<td>&nbsp;</td>
<tr>
<td>Polystyrene round-bottomed tubes with cell strainer caps (50 &mu;M pore size)</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352235</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of mouse salivary gland stem cells

Mature glandes salivaires d&#39;origine à la fois humains et de souris comprendre un minimum de cinq types de cellules, chacune d&#39;elles facilite la production et l&#39;excrétion de salive dans la cavité buccale. Cellules acineuses séreuses et muqueuses sont les protéines et les muqueuses des usines produisant de la glande, respectivement, et de représenter l&#39;origine de la production de salive. Une fois synthétisés, les différentes enzymatiques et d&#39;autres composants protéiques de la salive sont sécrétées par une série de cellules canalaires portant épithéliales de type morphologique, jusqu&#39;à l&#39;expulsion éventuelle de la salive à travers un conduit d&#39;importants dans la cavité de la bouche. La composition de la salive est également modifié par les cellules canalaires pendant ce processus. Dans la manifestation de maladies telles que le syndrome de Sjögren, et dans certaines situations cliniques telles que la radiothérapie pour les cancers de la tête et du cou, la production de salive par les glandes est considérablement réduite 1,2. La xérostomie qui en résulte, une sensation subjective de bouche sèche, affecte non seulement la capacité du patient à avaler et à parler, mais encourage également le développement de la carie dentaire et peut être socialement invalidante pour celui qui souffre. La restauration de la production de salive dans les conditions ci-dessus cliniques représente donc un besoin clinique non satisfait, et comme tel, plusieurs études ont démontré la capacité de régénération des glandes salivaires 3-5. Suite à l&#39;isolement des cellules souches, comme les populations de cellules de différents tissus au sein de la souris et le corps humains 6-8, nous avons montré en utilisant la méthode décrite que les cellules souches isolées à partir de glandes salivaires de souris peut être utilisée pour sauver la production de salive dans salivaires irradiées glandes 9,10. Cette découverte ouvre la voie pour le développement de base de cellules souches thérapies pour le traitement de la xérostomie conditions chez les humains, et aussi pour l&#39;exploration de la glande salivaire comme un microenvironnement des cellules contenant des multipotentes auto-renouvellement des capacités.

Watch this Scientific Journal Video about Isolation of Mouse Salivary Gland Stem Cells at JoVE.com

Isolation of Mouse Salivary Gland Stem Cells

Un protocole optimisé pour l&#39;isolement des cellules souches de la glande salivaire de souris est décrite. La méthode emploie digestion enzymatique et mécanique, et permet d&#39;isoler les cellules contenant salispheres avec des caractéristiques de cellules souches.

L&#39;isolement des cellules souches de la souris salivaires Gland

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

isolation-of-mouse-salivary-gland-stem-cells

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23.5K vues.  University Medical Center Groningen, University of Groningen. Un protocole optimisé pour l'isolement des cellules souches de la glande salivaire de souris est décrite. La méthode emploie digestion enzymatique et mécanique, et permet d'isoler les cellules contenant salispheres avec des caractéristiques de cellules souches.

Vidéo: Isolation of Mouse Salivary Gland Stem Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

L&#39;isolement et la transplantation de cellules souches hématopoïétiques (CSH)

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Biologie

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 &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Isolement et caractérisation de Hoechst Faible CD45 Négatifs Souris Cellules souches mésenchymateuses du poumon

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Isolement de cellules basales et les cellules sous-muqueux du canal de la glande de la souris Trachée

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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 ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Modification génétique et la recombinaison des cultures d&#39;organes salivaires Gland

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

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, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

L&#39;isolement, la culture, et la transplantation de cellules satellites du muscle

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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 ...