Les auteurs remercient Xiu-Ping Wang pour une aide initiale avec des cultures de DESC. Les auteurs sont financés en partie par des bourses et des subventions des Instituts nationaux de la santé (K99-DE022059 à AHJ, K12-GM081266 à MGC, K08-DE022377-02 à OH, et R01-DE021420 à ODK).

1. Disséquer Basse Hemi-mandibules de souris adulte <ol><li> Avant d&#39;effectuer ce protocole, obtenir l&#39;approbation institutionnel nécessaire et assurez-vous de respecter toutes les directives de protection des animaux. Euthanasier des animaux en utilisant la norme IACUC procédures approuvées. Pour ce travail asphyxie au CO2 a été utilisée, suivie par dislocation cervicale. <ol><li> OPTION: tête séparée du reste du corps en utilisant une lame de rasoir. </li></ol></li><li> Retirez la peau de la lèvre inférieure à l&#39;encolure. </li><li> Avec un scalpel, faire une incision entre les deux incisives inférieures, qui sont faiblement connectées. Après application d&#39;une pression, la mandibule est divisé en deux moitiés. </li><li> Caler le scalpel entre le condyle de la mâchoire et de l&#39;articulation mandibulaire temporelle et couper le long de la muscle de la mâchoire pour détacher la mâchoire. </li><li> Retirez tous les muscles, les tendons et les ligaments jusqu&#39;à ce qu&#39;il ne reste de l&#39;os. </li></ol> 2. Isoler l&#39;incisive <ol><li> Avec un scalpel # 15, Soigneusement raser l&#39;os à un angle de 45 ° à partir de l&#39;extrémité distale à l&#39;extrémité proximale de la région membraneuse. Utilisez la pointe du scalpel à prendre une distance n&#39;importe quel os restant, y compris les fragments d&#39;os sur le bord. Lentement commencer à chercher loin à la plaque corticale linguale de la mandibule, en commençant juste en dessous de la 3 ème molaire. </li><li> Continuez jusqu&#39;à ce que l&#39;incisive est propre, travailler soigneusement vers la zone contenant le CL. </li><li> Retirer nerf alvéolaire inférieur faisceau. </li><li> Faire une coupe propre pour enlever la première l&#39;os et les tissus à proximité de la CL. </li><li> Faire une deuxième coupe distale avec le scalpel, plus ou moins en dessous de la 2 ème molaire. La région incisive proximale est ensuite disséqué en insérant le scalpel entre l&#39;os et la surface interne de l&#39;incisive proximale. Ceci doit être effectué avec soin, de manière à ne pas déchirer le tissu. La motion de l&#39;incision est proximal-distal. STEP ALTERNATIVE: Retirer autant que possible os long de la zone au-dessus du incisor. Une fois la zone est dégagée, retirez soigneusement toute incisive en utilisant une taille de 5 pinces pour manipuler la partie de l&#39;émail plus proche de la région CL. </li><li> Placer le tissu disséqué (CL soit isolé comme dans la figure 3E, ou l&#39;ensemble de l&#39;incisive comme dans la variante Etape 2.5) dans 2% de collagénase dans du PBS 1X pendant 3-4 heures à 4 ° C dans une plaque de faible adhérence. Pour 1-10 incisives, utiliser 100 pi par puits dans une plaque de 12 puits. Pour plus de 10 incisives, utiliser 500 pi par puits dans une plaque à 6 puits à faible adhérence. </li></ol> 3. Microdissect la labiale col de l&#39;utérus boucle <ol><li> Retirer région CL de la collagénase et la place dans les médias DMEM/F12 froid. </li><li> Tirez doucement à l&#39;extrémité inférieure de la CL en utilisant soit la taille 5 forceps ou une seringue à insuline (1 cc, 28 G ½); mésenchyme va s&#39;écrouler tout l&#39;épithélium reste intacte. Le CL et l&#39;épithélium adjacent qui s&#39;étend latéralement comme une structure «en forme d&#39;aile&quot; seront visibles. </li><li> Supprimerle bourgeon apical de l&#39;épithélium adjacente en faisant une incision en forme de V sur chaque côté, et placer immédiatement en DMEM/F12 froid sur de la glace. </li><li> Répétez l&#39;opération pour tous les CL, de recueillir dans 1,5 ml tube de centrifugeuse. </li><li> Isoler tube de centrifugeuse avec des échantillons, retirer les supports, les remplacer par 100 ul solution de détachement cellulaire. </li><li> Incuber les tissus dans une solution de détachement des cellules pendant 30 min à 37 ° C. </li><li> Dissocier les tissus pour produire une suspension cellulaire unique par pipetage et l&#39;aide d&#39;un faible coefficient d&#39;adhérence pointe de la pipette 1000 pi vers le bas. Les cellules peuvent aussi être placées à travers un tamis cellulaire stérile pour réaliser la dissociation. </li><li> Compter les cellules avec un hémocytomètre, sur la plaque en matière plastique pour culture de tissus ou tout autre matériau souhaité. </li></ol> 4. Culture primaire de DESCs <ol><li> Cellules de la plaque dans une plaque à 6 puits à raison de 6.1 x 10 4 cellules / ml dans des milieux DESC, qui se compose de DMEM/F12 supplémenté avec de l&#39;EGF de la souris une protéine recombinante à une concentration de 20 ng / ml, FGF protéine recombinante à une concentration de 25 ng / ml, compléter 1X B27, et une solution à 1% d&#39;antibiotiques (pénicilline, 100 U / ml, streptomycine, 50 pg / ml). </li><li> Cultiver les cellules étalées sur une plaque à 6 puits à 37 ° C dans 5% de CO 2 dans 1 ml de DESC médias. Ne pas déranger cultures initiales pendant 5 jours après l&#39;étalement de permettre l&#39;adhésion cellulaire et la formation de colonies. </li><li> Pour le premier changement de médias, remplacer la moitié du volume (500 pi) de vieux médias avec la moitié du volume (500 pi) de nouveaux médias. Vérifiez colonies au microscope lors du changement de support. </li><li> Après le premier changement de support, continuer à changer de support (1-2 ml) tous les 2 jours. Vérifiez colonies au microscope lors du changement de support. </li></ol> 5. DESCs Passage <ol><li> Aspirer le milieu et laver les cellules 3x avec PBS préchauffé. </li><li> Ajouter préchauffé à 0,25% de trypsine-EDTA dans une solution saline et incuber à 37 ° C pendant 10 à 15 min pour les DESCs à détacher. </li><li> Ajouter un média DESC pour neutraliser la trypsine, la pipette doucement le long tissu plaque de culture pour recueillir les cellules, et les placer dans un tube de 15 ml conique. </li><li> Isoler les cellules à 800 g pendant 2 min. </li></ol>

<ol>
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Les auteurs n&#39;ont aucun conflit d&#39;intérêt à divulguer.

Les cellules épithéliales ont d&#39;abord été cultivées avec succès il ya 40 ans 21-24 et, plus récemment, réussi à isoler des cellules souches épithéliales 25-27 a fait progresser notre connaissance de la biologie épithéliale. Nous rapportons un protocole pour isoler le DESCs de l&#39;incisive de souris adulte, une population relativement peu étudié des cellules souches qui a le potentiel pour obtenir des informations importantes sur la biologie dentaire et la formation de l&#39;ém...

L&#39;une des caractéristiques uniques de vertébrés est l&#39;évolution de dents. La dent est devenue un important système modèle pour de nombreux domaines de recherche, comme les voies moléculaires et morphologiques spécialisations pertinentes à cet organe ont été étudiés à partir de plusieurs points de vue, y compris par les biologistes du développement et de l&#39;évolution 5. Plus récemment, le domaine de la médecine régénérative a commencé à acquérir de précieuses connaissances en utilisant la dent comme un modèle. En particulier, la découverte de cellules souches épithéliales dentaires a été un progrès important 6-13. Tous les rongeurs possèdent incisives croissance continue dont la croissance est alimentée par les cellules souches, faisant de ces dents d&#39;un système de modèle accessible pour étudier la régulation des cellules souches adultes. expériences en matière d&#39;étiquetage dans les années 1970 10,11, suivie par la lignée génétique des expériences de traçage 8,9,12,14, ont démontré que DESCs résident dans la région proximale de l&#39;incisive. La tigedescendance des cellules épithéliales dans le compartiment sur ​​le côté labial de déplacement hors de la niche putatif, connu sous le col de l&#39;utérus boucle labial (CL), et de contribuer à une population de cellules appelées transit d&#39;amplification (AT) des cellules (figure 1). Plus précisément, DESCs résident dans l&#39;épithélium externe de l&#39;émail (OEE) et réticulum étoilé (SR) 8,9,14, et l&#39;épithélium adamantin interne (EEI) donne lieu à des cellules d&#39;assistance technique qui progressent à travers un nombre limité de cycles cellulaires et mouvement de manière distale le long de la longueur de l&#39;incisive (Figure 1). Les améloblastes de différenciation dans l&#39;incisive de la souris continuent à se déplacer le long de distale de l&#39;incisive à un taux remarquable d&#39;environ 350 um en une seule journée 15,16. Comme ils se déplacent, les cellules se différencient en améloblastes matures et strate intermedium (SI) des cellules. Après le dépôt de la totalité de l&#39;épaisseur de la matrice de l&#39;émail, bon nombre des adamantoblastes subissent une apoptose, et les cellules restantes diminuer en taille et réguler la maturation émail &lt;sup&gt; 17. La lignée des autres types de cellules dans le CL labial, tels que le SR, est moins claire, et les données concernant les cellules souches dans le mésenchyme 18 et dans la face linguale CL commencent seulement à émerger. En utilisant le modèle de l&#39;incisive de la souris, un certain nombre de groupes ont travaillé à élucider les voies génétiques et les processus biologiques cellulaires impliqués dans le renouvellement des organes à base de cellules souches naturel. Cependant, la labiale CL contient un nombre relativement restreint de cellules, estimée à environ 5.000 par incisive de la souris, ce qui permet de travailler avec des cellules primaires contester. Par conséquent, des efforts ont été faits pour la culture et d&#39;élargir ces cellules in vitro 6,16,19,20 afin d&#39;ouvrir de nouvelles portes à des approches expérimentales qui ne sont pas réalisables in vivo. L&#39;étude la plus récente a démontré que ces cellules peuvent à la fois se renouveler et de se différencier en cellules exprimant amelogenin lorsqu&#39;elles sont cultivées 13. Ici, nous décrivons et démontrons une méthode pour ee culture fiable et cohérente des cellules de la labiale de la souris CL.

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			<td height="63" style="height:63px;width:216px;">Forceps, size 5</td>
			<td style="width:83px;">Fine Science Tools</td>
			<td style="width:119px;">11251-30</td>
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isolation and culture of dental epithelial stem cells from the adult mouse incisor

Comprendre les mécanismes cellulaires et moléculaires qui sous-tendent la régénération de la dent et le renouvellement est devenu un sujet de grand intérêt 1-4, et l&#39;incisive de la souris fournit un modèle pour ces processus. Cet organe remarquable pousse en permanence tout au long de la vie de l&#39;animal et génère tous les types de cellules nécessaires de piscines actives de cellules souches adultes logés dans le labiale (vers la lèvre) et multilingue (vers la langue) boucle col de l&#39;utérus (CL) des régions. Seules les cellules souches de l&#39;art dentaire labiale CL donnent lieu à adamantoblastes qui génèrent émail, l&#39;enveloppe externe de dents, à la surface labiale. Cette formation de l&#39;émail asymétrique permet à l&#39;abrasion, à la pointe de l&#39;incisive, et les progéniteurs et les cellules souches dans le incisive proximale veiller à ce que les tissus dentaires sont constamment renouvelés. La capacité d&#39;isoler et de cultiver ces cellules progénitrices ou souches in vitro permet leur expansion et ouvre les portes à de nombreuses expériences pas réalisables in vivo, comme htests de débit aute de facteurs de régulation de cellules souches potentiel. Ici, nous décrivons et démontrons une méthode fiable et cohérente à des cellules de culture de la CL vestibulaire de l&#39;incisive de la souris.

L&#39;hémi-mandibule de la souris un comprend croissance continue incisive et trois molaires enracinées (figure 1A). Toutes les dents sont constituées de la dentine et de l&#39;émail, les deux composants minéralisées de la couronne de la dent (Figures 1A et 1A &#39;). Les maisons des incisives deux niches de cellules souches appelées CL labiale et linguale CL, et l&#39;émail est formé exclusivement sur ​​le côté labial (figure 1A). DESCs ...

Watch this Scientific Journal Video about Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor at JoVE.com

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

L&#39;incisive de la souris en croissance continue fournit un modèle pour l&#39;étude de renouvellement des tissus dentaires à partir de cellules souches épithéliales dentaires (DESCs). Un système solide de façon cohérente et fiable obtention de ces cellules de l&#39;incisive et leur expansion in vitro est rapporté ici.

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 ...

isolation-culture-dental-epithelial-stem-cells-from-adult-mouse

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JoVE Journal

Biology

15.0K vues.  University of California, San Francisco.  L'incisive de la souris en croissance continue fournit un modèle pour l'étude de renouvellement des tissus dentaires à partir de cellules souches épithéliales dentaires (DESCs). Un système solide de façon cohérente et fiable obtention de ces cellules de l'incisive et leur expansion in vitro est rapporté ici. 

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

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 ...

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Biologie

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolement et culture des fibres musculaires individuelles et de leurs cellules satellites du muscle squelettique adulte

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve cellules souches adultes Isolation de cultures de fibroblastes humains primaires

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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 Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolement, caractérisation et différenciation des cellules souches cardiaques du coeur de souris adulte

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

Isolement et culture simultanés des myocytes auriculaires, des myocytes ventriculaires et des non-myocytes provenant d’un cœur de souris adulte

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolement et culture de kératinocytes oraux primaires du palais de souris adulte

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...