1. Regent Vorbereitung <ol><li> Buffer: 1% (w / v) Rinderserumalbumin in balanced salt Hank-Lösung </li><li> Rekonstituieren Enzyme. Hyaluronidase-Enzym: 40 mg / mL, in Puffer gelöst. Collagenase II: 23 mg / mL, in Puffer gelöst. Verwenden Sie frisch zubereitete Enzymlösungen frisch für jede Isolation. Wenn aufgelöst, Lagerung bei 4 ° C bis zur Verwendung für die Verdauung. </li><li> 50 mM Calciumchlorid in destilliertem Wasser. Filter durch einen 0,2 uM Filter mit einer Porengröße zu sterilisieren. </li><li> Maus Speicheldrüse (MSG) Kulturmedium: DMEM: F12 mit Penicillin (100 IU / mL), Streptomycin (100 ug / mL), Glutamax (2 mM), epidermal growth factor-2 (20 ng / mL), Fibroblasten-Wachstumsfaktor -2 (20 ng / mL), N2 gegen Aufpreis (1%), Insulin (10 ug / ml) und Dexamethason (1 pM). </li></ol> 2. Mechanische und enzymatische Verdauung Tissue <ol><li> Wiegen Sie die seziert Speicheldrüsen. </li><li> Hacken Drüsen zu einem homogenen Brei mit sterilen gebogenen Dissektion Schere in einer kleinen Petrischale. </li><li> Sammeln Sie zerkleinerten Gewebes in 14 ml-Röhrchen mit 1 ml Puffer pro 80mg submandibularis Gewebe. Spülen Sie die Petrischalen sauber von Gewebe mit einigen der Puffer. </li><li> Add another 1 ml Puffer pro 80 mg Gewebe, von 25 ul Kollagenase II Enzymlösung, 25 ul Hyaluronidase Enzymlösung und 250 ul Calciumchlorid-Lösung pro 80 mg Gewebe gefolgt. Wenn die Arbeit mit großen Mengen von Gewebe, Schritte von 2,4 bis 2,9 in T25 Zellkulturflaschen für die Bequemlichkeit durchgeführt werden kann. </li><li> Inkubieren in einem Schüttel-Wasserbad bei 37 ° C für 20 Minuten. Entfernen Sie Rohre und verreiben mit einer Pipette auf Enzym gründlich mischen durch das Gewebe wieder. </li><li> Ersetzen Sie im Wasserbad für weitere 20 Minuten. </li><li> Sammeln Gewebe durch Zentrifugation bei 400 xg für 8 Minuten. Überstand. </li><li> Resuspendieren in 2 ml Puffer für je 80 mg Gewebe, und wiederholen Enzym und Calciumchlorid zusätzlich wie oben beschrieben. Inkubieren 20 Minuten in Schüttelwasserbad. Entfernen Sie Rohre und verreiben durch eine Pipette, um Enzyme gründlich zu mischen. </li><li> Inkubieren letzten 20 min in Schüttelwasserbad. Sammeln Sie die Zellen durch Zentrifugation wie oben, Überstand verwerfen. </li></ol> 3. Waschschritte <ol><li> Resuspendieren je 80 mg Gewebe in 2 mL Puffer und Pipette, um Gewebe frei von Enzymen zu waschen. </li><li> Centrifuge wie zuvor zu sammeln. Überstand. </li><li> Wiederholen Sie waschen mit 1 mL Puffer pro 80 mg Gewebe. Centrifuge zu sammeln, Überstand verwerfen. </li></ol> 4. Filtering <ol><li> Resuspendieren Gewebe Lösung in 1 mL Puffer pro 80 mg Gewebe. </li><li> Add-Lösung bis 100 um Porengröße Filter über 50 mL Falcon-Röhrchen platziert. Nicht mehr als 3 ml zerkleinerte Gewebe Lösung pro Spalte, die als Filter blockiert werden. Lassen Sie die durchsickern. Entfernen Sie gefilterte Material hängen an der Unterseite des Filters durch eine Pipette, und fügen Sie Filtrat. </li><li> Verwenden Spritze mit 26-Gauge-Nadel, um Filtrat von 50ml Röhrchen nehmen und gelten bis 50 Mikrometer Porengröße Filter auf 5 ml Tuben. Lassen Sie die Filter, durch, unterstützt durch Lösen Deckel, wenn nötig. </li><li> Zentrifugenröhrchen wie zuvor zu sammeln. Überstand. </li></ol> 5. Plating-und Mittelbetriebe <ol><li> Kombinieren Sie alle Pellets in einem Band. Graf mit automatisierten Zelle Zähler oder Hämocytometer. </li><li> Platte Zellen bei einer Dichte von 0,4 x 10 6 Zellen pro Vertiefung einer 12-Well-Platte, oder 2,67 x 10 6 Zellen pro T25 Zellkulturflasche. 1 ml MSG Medium in jede Vertiefung oder 6 ml in jedes T25 Kolben. </li><li> Inkubation bei 37 ° C. Spheres muss deutlich sichtbar von Tag 2. </li></ol> 6. Repräsentative Ergebnisse: Nach zwei Minuten vor drei Tagen in Kultur werden kleine Aggregate von Zellen (salispheres) werden in den Kulturen deutlich. Salispheres wird weiterhin in der Größe über einen Zeitraum von zehn Tagen in Kultur zu züchten. Vertreter Phasenkontrast-Mikroskopie-Aufnahmen von salispheres sind in Abbildung 1 dargestellt. Proliferierenden Zellen, die Stammzellen-assoziierte Markerproteine ​​können aus diesen Bereichen isoliert werden, optimal zwischen Tage 3-5 post Isolation und sind in der Lage Differenzierung in funktionale, Speichel produzieren Azinuszellen. <img src="/files/ftp_upload/2484/2484fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Salisphere Bildung in vitro. Nach der mechanischen und enzymatischen Aufschluss über die vorliegende Protokoll, können Kugeln mit zunehmender Größe in der schwimmenden Kulturen gefunden werden. Panels sind repräsentativ für die Phasenkontrast-Mikroskopie Bilder von Kugeln von Tagen 0 (A), 4 (B), 7 (C) und 10 (D). Balken = 50 um.

<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>

Die Gewebekultur hier beschriebene Methode stellt eine reproduzierbare Protokoll für die Isolierung von Stammzellen mit salispheres aus den Speicheldrüsen der Mäuse. Studien unter Verwendung von Zellen auf diese Weise isoliert wurden, die Regenerationsfähigkeit der Speicheldrüse Stammzellen Zellen 9 hervorgehoben. Die Transplantation von hundert von c-Kit +-Zellen aus dem salispheres abgeleitet induzierte funktionelle Erholung von bestrahlten Maus Speicheldrüsen. Diese Daten sind spannend und bieten einen Ausgangspunkt für die Untersuchung von Stammzell-Therapie für Xerostomie. Viele Wege bleiben jedoch untersucht werden, einschließlich der vollständigen Markerprotein Expressionsprofil der Stammzellen, die Fähigkeit der submandibularis, um die Funktion der bestrahlten Parotis Speicheldrüsen und umgekehrt zu retten, und die Charakterisierung der putativen in vivo Stammzellnische der der Zellen. Letztlich ist die Übersetzung dieses Protokoll für die menschliche Gewebeproben und die anschließende Potenzial für die Therapie der Xerostomie bei menschlichen Patienten unter Verwendung der isolierten Zellen der aufregendsten Anwendung der beschriebenen Methode.

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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

Ältere Speicheldrüsen von Mensch und Maus Herkunft aus einem Minimum von fünf Zelltypen, von denen jede die Produktion und Ausschüttung von Speichel erleichtert in die Mundhöhle. Serösen und Schleimhäuten Azinuszellen sind die Protein-und Schleim produzieren Fabriken der Drüse bzw. und stellen den Ursprung der Speichelproduktion. Sobald synthetisiert werden die verschiedenen enzymatischen und anderen proteinhaltigen Komponenten des Speichels durch eine Reihe von duktalen Zellen, die epithelial-Art-Morphologie abgesondert wird, bis die eventuellen Ausweisung der Speichel durch einen großen Kanal in der Mundhöhle. Die Zusammensetzung des Speichels wird auch durch den duktalen Zellen während dieses Prozesses verändert. In der Manifestation von Krankheiten wie Sjögren-Syndrom, und in einigen klinischen Situationen wie Strahlentherapie bei Kopf-Hals-Tumoren, die Speichelproduktion durch die Drüsen ist dramatisch 1,2 reduziert. Die daraus resultierenden Xerostomie, ein subjektives Gefühl der Mundtrockenheit, betrifft nicht nur die Fähigkeit des Patienten zu schlucken und sprechen, sondern fördert auch die Entwicklung von Karies und kann sozial schwächenden für den Erkrankten. Die Wiederherstellung der Speichel-Produktion in den oben genannten klinischen Bedingungen stellt daher einen ungedeckten klinischen Bedarf, und als solche haben mehrere Studien die Regenerationsfähigkeit der Speicheldrüsen 3-5 gezeigt. Im Anschluss an die Isolierung von Stammzellen, wie Populationen von Zellen aus verschiedenen Geweben innerhalb der Maus und menschlichen Körpern 6-8, haben wir gezeigt, mit dem beschriebenen Verfahren, dass Stammzellen aus der Maus Speicheldrüsen isoliert verwendet werden, um die Speichelproduktion in bestrahlten Speicheldrüsen zu retten Drüsen 9,10. Diese Entdeckung ebnet den Weg für die Entwicklung von Stammzell-basierten Therapien für die Behandlung von Xerostomie Verhältnisse beim Menschen, aber auch für die Erforschung der Speicheldrüse als Mikroumgebung enthaltenden Zellen mit multipotenten selbst erneuernde Fähigkeiten.

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

Isolation of Mouse Salivary Gland Stem Cells

Ein optimiertes Protokoll für die Isolierung von Stammzellen aus der Maus Speicheldrüse beschrieben. Die Methode nutzt enzymatische und mechanische Verdauung, und erlaubt Isolierung salispheres enthaltenden Zellen mit Eigenschaften von Stammzellen.

Isolierung von Maus Speicheldrüsen Stem Cells

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

Research

JoVE Journal

Biology

23.6K Aufrufe.  University Medical Center Groningen, University of Groningen. Ein optimiertes Protokoll für die Isolierung von Stammzellen aus der Maus Speicheldrüse beschrieben. Die Methode nutzt enzymatische und mechanische Verdauung, und erlaubt Isolierung salispheres enthaltenden Zellen mit Eigenschaften von Stammzellen.

Serie: Isolation of Mouse Salivary Gland Stem Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

Isolierung und Transplantation von hämatopoetischen Stammzellen (HSZ)

Forschung

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.

Isolation und Ableitung von embryonalen Maus-Keimzellen

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.

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 ...

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 & 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.

Isolation und Charakterisierung von Hoechst Low CD45 Negative Mouse Lung mesenchymalen Stammzellen

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.

Isolierung von Basalzellen und Submuköse Gland Duct Zellen aus Maus 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. ...

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 ...

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.

Genetic Modification und Rekombination von Speicheldrüsen Organkulturen

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.

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, 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;

Isolierung, Kultur und Transplantation von Muskel-Satelliten-Zellen

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.

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 ...