Die Autoren danken Xiu-Ping Wang für die erste Hilfe bei DESC Kulturen. Die Autoren sind zum Teil durch Stipendien und Zuschüsse aus den National Institutes of Health (K99-DE022059 zu AHJ, K12-GM081266 MGC, K08-DE022377-02 zu OH-und R01-DE021420 zu ODK) finanziert.

1. Dissect Nieder Hemi-Mandibeln von erwachsenen Maus <ol><li> Vor der Durchführung dieses Protokolls erforderlichen institutionellen Genehmigung zu erhalten und sicher sein, mit allen Tierpflege-Richtlinien. Euthanize Tier mit Standard IACUC genehmigten Verfahren. Für diese Arbeit CO 2-Erstickung wurde verwendet, gefolgt durch zervikale Dislokation. <ol><li> OPTIONAL: Separate Kopf vom Rest des Körpers mit einer Rasierklinge. </li></ol></li><li> Entfernen Sie die Haut vor der Unterlippe an den Halsausschnitt. </li><li> Mit einem Skalpell einen Einschnitt zwischen den beiden unteren Schneidezähne, die lose miteinander verbunden sind. Beim Anlegen von Druck wird der Unterkiefer in zwei Hälften geteilt werden. </li><li> Keil das Skalpell zwischen dem Gelenkkopf des Kiefers und der Kiefergelenk und neben der Kiefermuskel geschnitten, um den Kiefer zu lösen. </li><li> Entfernen Sie das gesamte Muskel-, Sehnen-und Bänder bis nur noch die Knochenreste. </li></ol> 2. Isolieren Sie die Schneidezahn <ol><li> Mit einem Skalpell Nr. 15Sorgfältig rasieren den Knochen in einem Winkel von 45 ° von dem distalen Ende zu dem proximalen Ende des membranartigen Bereichs. Verwenden Sie die Spitze des Skalpells zu holen Sie verbleibende Knochen einschließlich der Knochenfragmente am Rand. Langsam beginnen, bei der lingualen kortikalen Platte des Unterkiefers entfernt holen, beginnend knapp unterhalb der 3. Molaren. </li><li> Fortfahren, bis der Schneidezahn ist sauber, sorgfältig arbeiten auf den Bereich, der die CL. </li><li> Entfernen alveolaris inferior Nervenbündel. </li><li> Machen Sie einen sauberen Schnitt, zuerst die Knochen-und Gewebe proximal der CL zu entfernen. </li><li> Machen Sie einen zweiten Schnitt distal mit dem Skalpell, etwa unter dem 2. Molaren. Das proximale Schneidezahnbereich wird dann durch Einsetzen des Skalpells zwischen dem Knochen und der medialen Fläche des proximalen Schneidezahn präpariert. Dies muß sorgfältig durchgeführt werden, um nicht in das Gewebe zu zerreißen. Der Schnitt Bewegung ist proximal-distalen. ALTERNATIVE STEP: Zu entfernen, so viel Knochen wie möglich an dem Bereich über dem incisor. Sobald Bereich wird gelöscht, entfernen Sie vorsichtig gesamte Schneidezahn mit eine Größe von 5 Zange, um den Zahnschmelz Teil hand am nächsten CL Region. </li><li> Platzieren seziert Gewebe (entweder isoliert CL, wie in Fig. 3E oder ganze Schneidezahn nach alternativen Schritt 2.5) in 2% Kollagenase in 1X PBS für 3-4 Stunden bei 4 ° C in einer Niederhalten Platte. Für 1-10 Schneidezähne, verwenden Sie 100 ul pro Well in einer 12-Well-Platte. Für mehr als 10 Schneidezähne, verwenden Sie 500 ul pro Well in einer 6-Well Platte geringe Haftung. </li></ol> 3. Microdissect die Labial Cervical-Schleife <ol><li> Entfernen CL Region aus der Kollagenase und in kaltem DMEM/F12-Medium. </li><li> Ziehen Sie am unteren Ende der CL entweder mit der Größe 5 Zange oder einer Insulinspritze (1 ml, 28 G ½); das Mesenchym auseinander fallen, während das Epithel intakt bleibt. Die CL-und die benachbarte Epithel, die sich seitlich als &quot;flügelartigen&quot; Struktur erstreckt sichtbar. </li><li> Entfernendie apikalen Knospe von der benachbarten Epithel, indem ein V-förmiger Einschnitt auf jeder Seite, und sofort in kaltem DMEM/F12 auf Eis. </li><li> Wiederholen Sie für alle CLs, sammeln in 1,5 ml Mikrozentrifugenröhrchen. </li><li> Spin down Mikrozentrifugenröhrchen mit Proben, entfernen Medien, ersetzen mit 100 ul Zellablösung Lösung. </li><li> Gewebe in Zellentfernungslösung für 30 min bei 37 ° C. </li><li> Distanzieren das Gewebe, um eine Einzelzellsuspension durch vorsichtiges Auf-und Abpipettieren mit einer 1.000 ul geringer Haftung Pipettenspitze herzustellen. Zellen können auch durch ein steriles Sieb Zelle angeordnet werden, um die Dissoziation zu erreichen. </li><li> Zählen von Zellen mit Hämozytometers, Platte auf Gewebekultur Kunststoff oder anderen gewünschten Material. </li></ol> 4. Primärkultur von DESC <ol><li> Platten Zellen in einer 6-Well-Platte bei 1-6 × 10 4 Zellen / ml in DESC Medien, die von DMEM/F12 mit Maus-EGF rekombinantes Protein ergänzt war, bei einer Konzentration von 20 n bestehtg / ml FGF rekombinantes Protein in einer Konzentration von 25 ng / ml, 1 × B27 Ergänzung und 1% Antibiotika-Lösung (Penicillin, 100 U / ml Streptomycin, 50 ug / ml). </li><li> Wachsen Zellen auf einer 6-Well-Platte ausplattiert, auf 37 ° C in 5% CO 2 in 1 ml DESC Medien. Anfangs Kulturen für 5 Tage nach der Beschichtung, um die Zellhaftung und Koloniebildung ermöglichen nicht stören. </li><li> Zum ersten Medienwechsel, ersetzen Sie die Hälfte des Volumens (500 ul) alten Medien mit Halb Volumen (500 ul) der neuen Medien. Überprüfen Kolonien unter dem Mikroskop beim Medienwechsel. </li><li> Nach dem ersten Medienwechsel, weiterhin Medien (1-2 ml) alle 2 Tage ändern. Überprüfen Kolonien unter dem Mikroskop beim Medienwechsel. </li></ol> 5. Passage DESC <ol><li> Saugen Medien und waschen Sie die Zellen 3x mit vorgewärmter PBS. </li><li> Hinzufügen vorgewärmten 0,25% Trypsin-EDTA in einer Salzlösung und Inkubation bei 37 ° C für 10-15 Minuten für die DESC zu lösen. </li><li> In DESC Medien, um das Trypsin zu neutralisieren, vorsichtig pipettieren entlang Gewebekulturplatte, um die Zellen, und in eine 15 ml konischen Röhrchen zu sammeln. </li><li> Spin down Zellen bei 800 g für 2 min. </li></ol>

<ol>
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Die Autoren haben keine Interessenkonflikte offen zu legen.

Epithelzellen wurden zuerst erfolgreich kultiviert vor 40 Jahren über 21-24, und in jüngerer Zeit erfolgreiche Isolierung von epithelialen Stammzellen 25-27 unserem Wissen epithelialer Biologie fortgeschritten. Wir berichten über ein Protokoll zur Isolierung der DESC der erwachsenen Maus Schneidezahn, eine relativ wenig erforschten Population von Stammzellen, die das Potenzial, wichtige Einblicke in die Biologie und Zahnschmelzbildung ergeben hat. Dieses Protokoll wurde zunächst auf früheren B...

Eines der einzigartigen Merkmale von Wirbeltieren ist die Entwicklung der Zähne. Der Zahn ist ein wichtiges Modellsystem für viele Bereiche der Forschung, da die molekularen Signalwege und relevant zu dieser Orgel morphologischen Spezialisierungen aus verschiedenen Perspektiven untersucht worden, unter anderem durch Entwicklungs-und Evolutionsbiologen 5. Vor kurzem hat der Bereich der regenerativen Medizin hat damit begonnen, wertvolle Erkenntnisse zu gewinnen mit dem Zahn als Modell. Insbesondere die Entdeckung von Zahn epithelialen Stammzellen ist ein wichtiger Fortschritt 6-13. Alle Nager besitzen stetig wachsenden Schneidezähne, deren Wachstum durch Stammzellen angeheizt, so dass diese Zähne ein zugängliches Modellsystem, um die adulte Stammzellregulation zu studieren. Markierungsexperimente in der 1970er 10,11, gefolgt von genetischer Abstammung Tracing 8,9,12,14 Experimente haben gezeigt, dass DESC befinden sich im proximalen Bereich der Schneidezähne. Der SchaftZelle Nachkommen in der epithelialen Fach auf der labialen Seite bewegen sich aus der vermeintlichen Nische, wie die Lippen-Hals-Schleife (CL) bekannt ist, und zu einer Population von Zellen, die sogenannten Transit-Verstärkung (TA)-Zellen (Abbildung 1). Genauer gesagt, befinden DESC in der äußeren Schmelzepithels (OEE) und sternförmigen Retikulum (SR) 8,9,14 und die innere Schmelzepithels (IEE) gibt Anlass zu den TA-Zellen, die durch eine begrenzte Anzahl von Zellzyklen fortschreiten und dann bewegen distal entlang der Länge der Schneidezähne (Abbildung 1). Die Differenzierung Ameloblasten in der Maus Schneidezahn weiter distal entlang der Schneidezähne mit einer bemerkenswerten Geschwindigkeit von etwa 350 um an einem einzigen Tag 15,16 bewegen. Wie sie sich bewegen, zu differenzieren die Zellen zu reifen Ameloblasten und Stratum intermedium (SI)-Zellen. Nach dem Aufbringen der gesamten Dicke von Schmelzmatrix, viele der Adamantoblasten Apoptose, und die verbleibenden Zellen in der Größe schrumpfen und zu regulieren Schmelzreifung &lt;sup&gt; 17. Die Linie der anderen Zelltypen im Lippen CL, wie der SR, ist weniger klar, und Daten über den Stammzellen im Mesenchym 18 und in der lingualen CL beginnen erst zu entstehen. Mit der Maus Schneidezahn-Modell, eine Reihe von Gruppen gearbeitet haben, um die genetischen Signalwege und zellbiologische Prozesse in natürlichen Stammzellen-basierten Orgelerneuerung beteiligt aufzuklären. Jedoch enthält der labialen CL eine relativ kleine Anzahl von Zellen, auf etwa 5000 pro Maus Schneidezahn, die die Arbeit mit primären Zellen Herausforderung macht. Daher haben sich bemüht, Kultur wurden und erweitern diese Zellen in vitro 6,16,19,20, um neue Türen zu experimentellen Ansätzen, die nicht in vivo erreichbar sind zu öffnen. Die jüngste Studie hat gezeigt, dass diese Zellen sowohl selbst erneuern und zu differenzieren in amelogenin exprimierenden Zellen kultiviert, wenn 13. Hier beschreiben wir und zeigen ein Verfahren zur the zuverlässige und konsistente Kultur von Zellen von der Maus labialen 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>
			<td style="width:167px;">FST by Dumont, Dumoxel,&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Forceps Straight, fine, 3.5</td>
			<td style="width:83px;">Roboz</td>
			<td style="width:119px;">RS5070</td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">Razorblades</td>
			<td style="width:83px;">Electron Microscopy Services</td>
			<td style="width:119px;">#71960</td>
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			<td height="21" style="height:21px;width:216px;">Scalpel Handle No.3</td>
			<td style="width:83px;">VWR</td>
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			<td></td>
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			<td height="63" style="height:63px;width:216px;">Feather Blade No. 15</td>
			<td style="width:83px;">Electron Microscopy Services</td>
			<td style="width:119px;">#72044-15</td>
			<td>Surgical Stainless Steel</td>
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			<td height="126" style="height:126px;width:216px;">Collagenase Type-1 filtered</td>
			<td style="width:83px;">Worthington Biochemical Corporation</td>
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			<td height="21" style="height:21px;width:216px;">Insulin syringe</td>
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			<td height="42" style="height:42px;width:216px;">Accumax</td>
			<td style="width:83px;">EMD Millipore</td>
			<td style="width:119px;">#SCR006</td>
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			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:83px;">Gibco</td>
			<td style="width:119px;">11320-033</td>
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			<td height="21" style="height:21px;width:216px;">EGF</td>
			<td style="width:83px;">R&#38;D</td>
			<td style="width:119px;">2028-EG-200</td>
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			<td height="21" style="height:21px;width:216px;">FGF2</td>
			<td style="width:83px;">R&#38;D</td>
			<td style="width:119px;">233-FB-025</td>
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</table>

isolation and culture of dental epithelial stem cells from the adult mouse incisor

Das Verständnis der zellulären und molekularen Mechanismen, die den Zahnwiedergeburt und Erneuerung zugrunde liegen, hat sich zu einem Thema von großem Interesse 1-4, und die Maus Schneidezahn ein Modell für diese Prozesse. Diese bemerkenswerte Orgel wächst kontinuierlich während des ganzen Lebens des Tieres und alle notwendigen Zelltypen aus dem aktiven Pools von adulten Stammzellen in der labialen (in Richtung der Lippe) und lingual (zur Zunge) Hals-Schleife (CL) untergebracht Regionen erzeugt. Nur die Zahn Stammzellen von der labialen CL verursachen Adamantoblasten die Emaille, die äußere Ummantelung der Zähne, auf der labialen Oberflächen zu erzeugen. Diese asymmetrische Zahnschmelzbildung ermöglicht Abrieb an der Schneidezahnspitze und Vorläuferzellen und Stammzellen in der proximalen Schneidezahn sicherzustellen, dass die Zahngewebe werden ständig aufgefüllt. Die Fähigkeit, in vitro zu isolieren und zu wachsen diese Vorläufer-oder Stammzellen erlaubt die Expansion und öffnet Türen zu zahlreichen Versuchen nicht erreichbar in vivo, wie Hoch Durch Prüfung von potenziellen Stammzellregulationsfaktoren. Hier beschreiben wir und zeigen eine zuverlässige und konsistente Methode zur Kultivierung von Zellen von der labialen CL der Maus Schneidezahn.

Die Maus halbKiefers weist einen kontinuierlich wachsenden Schneidezähne und Backenzähne drei Wurzeln (Fig. 1A). Alle Zähne aus Dentin und Zahnschmelz, der zwei mineralisierten Komponenten der Zahnkrone (Fig. 1A und 1A &#39;). Die Schneidezähne beherbergt zwei Stammzellnischen genannt labialen und lingualen CL CL und Email wird ausschließlich auf der labialen Seite (Abbildung 1A &#39;) gebildet wird. DESC für Schneidezahn Schmelzbildung verantwort...

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

Das kontinuierlich wachsende Maus Schneidezahn stellt ein Modell für die Untersuchung Erneuerung der Zahngewebe aus Zahn epithelialen Stammzellen (DESC). Ein robustes System für konsistent und zuverlässig diese Zellen von den Schneide erhalten und in vitro expandiert sie hier berichtet wird.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

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

Research

JoVE Journal

Biology

15.0K Aufrufe.  University of California, San Francisco.  Das kontinuierlich wachsende Maus Schneidezahn stellt ein Modell für die Untersuchung Erneuerung der Zahngewebe aus Zahn epithelialen Stammzellen (DESC). Ein robustes System für konsistent und zuverlässig diese Zellen von den Schneide erhalten und in vitro expandiert sie hier berichtet wird. 

Serie: 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.

Isolierung von Retinal Stammzellen aus der Maus 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 ...

Forschung

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.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

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.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

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.

Isolation und Kultur der einzelnen Muskelfasern und ihre Satelliten aus adulten Skelettmuskel

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.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

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.

Induktion und Analyse von epithelialen zu mesenchymalen 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.

Isolierung und Kultur der primären Maus Keratinozyten aus neonatalen und erwachsenen Maus Haut

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.

Isolierung, Charakterisierung und Differenzierung von kardialen Stammzellen von Erwachsenen Maus Herzen

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.

Gleichzeitige Isolierung und Kultur von Atrial Myozyten, ventrikulären Myozyten und Nicht-Myozyten aus einem erwachsenen Mausherz

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.

Isolierung und Kultur von primären oralen Keratinozyten aus dem erwachsenen Mausgaumen

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