Die Autoren sind dankbar für die Frau Haiying Pan und Herr Ian Bellayr für die technische Hilfe auf Zellisolation für den aktuellen Filmen und Mr. Kyle Holden und Mr. Jonathan Lucas für ihre Hilfe auf Folie bearbeiten. Vielen Dank an die folgende Liste von Personen, die mit der Entwicklung der modifizierten Vorbeschichtungszyklus Techniken in den letzten Jahren beteiligt waren: Drs. Burhan Gharaibeh, Baohong Cao, Deasy Bridget, Thomas R. Payne, Aiping Lu, Hairong Peng, Hideki Oshima, Bo Zeng, Xiaodong Mu, Kimimasa Tobita, Ron Jankowski, Makato Ikezawa. Wir sind dankbar für die technische Unterstützung von James H. Cummins, Ryan Pruchnic, Joseph Feduska, Rebecca C. Schugar, Haiying Pan und Jessica Tebbets. Der Autor möchte auch die finanzielle Unterstützung für diese Arbeit von der Gesellschaft für Muskeldystrophie (MDA), der National Institutes of Health (NIH), das Department of Defense (DOD), dem Kinderkrankenhaus von Pittsburgh von UPMC, das Department of anerkennen Orthopädische Chirurgie und der University of Pittsburgh.

In der Regel erfordert die vollständige geändert Vorbeschichtungszyklus Verfahren 1-2 Tage Vorbereitung, 1 Woche für die Durchführung der technischen Verfahren, 2-3 Tage der Zelle Identifikation mit Immunzytochemie, und eine zusätzliche 2-3 Wochen von Stammzellen Bevölkerung Expansion. Die Ausrüstung für die Stammzell-Kultur erforderlich sind, ähnlich wie bei anderen Zellkulturen, die eine Tischzentrifuge, CO 2-Inkubator, Laminar Flow Gewebekultur Kapuze und einem inversen Mikroskop mit Fluoreszenz-und einer digitalen Kamera ausgestattet sind. Die isolierten Stammzellen können auch geklont werden können und langfristig aufbewahrt in flüssigem Stickstoff für die aktuelle und zukünftige Studien 1,2 sein. Alle Reagenzien und Materialien für dieses Verfahren verwendet werden, müssen steril sein und behandelt aseptisch. Schritt 1: Vorbereitung <ol><li> Collagen Mantel Gewebekulturschalen und Flaschen: Typ I Kollagen aus Kalbsleder (0,1 g in 1 Liter; Sigma-Aldrich) abgeleitet. Die Kunststoffprodukte zu beschichten sind: T-25 Flaschen, 6 oder 12-Well-Platten, Geschirr (60 und 100 mm, BD Falcon) nach wie vor 1,2 beschrieben. Schütteln Sie die Kunststoffprodukte jede halbe Stunde, 4 Stunden auf eine gleichmäßige Beschichtung des Kollagens zu gewährleisten. Das Kollagen wird dann entfernt. Und die beschichtete Kunststoffprodukte ist dann erlaubt, in der Gewebekultur Kapuze mit dem UV-Licht und die Haube sowohl auf trockenen, um die Sterilität zu gewährleisten, und schließlich wieder verschlossen oder abgedeckt für eine spätere Verwendung. </li><li> Bereiten Zellkulturmedium: 400ml DMEM (DMEM, hohe Glukose; Invitrogen), 50 mL Hitze inaktiviertes fötales Rinderserum (FBS, Invitrogen), 50 mL Pferdeserum (HS, Invitrogen) und 5 mL Hühnerembryo-Extrakt (CEE; Accurate Chemical Co. ) wird im Vakuum filtriert mit einem Einweg 0,22-um 500-ml sterile Filter-System (Corning) werden. Sterile Penicillin / Streptomycin-Lösung (Invitrogen) wird schließlich 100 Einheiten / ml zugesetzt werden. Die hergestellten Lösungen können bei 4 ° C für mindestens einen Monat gelagert werden. Diese Lösung wird verwendet, um die Zellen aus der Flasche lösen beim Aufteilen Kulturen oder der Vorbereitung der Zellen zur Transplantation. </li><li> Warm up-Lösungen für die Zell-Trennung: Die folgenden Reagenzien müssen bis zu 37 ° C erwärmt werden vor ihrem Einsatz für die Zell-Trennung: Hanks Salzlösung (HBSS, Invitrogen), 0,2% (wt / vol) Kollagenase Typ XI (Sigma -Aldrich), mit einem Durchschnitt von 3.200 Kollagen Verdauung Einheiten pro ml HBSS; Dispase 2,4 units/1mL (Invitrogen), 0,5% (wt / vol) Trypsin (Invitrogen). </li><li> Sterilisieren chirurgische Geräte in Vorbereitung: Chirurgischer Eingriff erfordern sterile OP-Instrumente, einschließlich: unterschiedlich große Zange, Mikro-Schere, Schere Iris und / oder Skalpellklingen. Zusätzlich, 15 und 50-mL Polypropylen-Zentrifugenröhrchen (BD Falcon); Zellsieb (Porengröße 70 m) (BD Falcon), 18 -, 23 - und 27-Gauge-Nadeln (BD PrecisionGlide) und 10 - oder 20-ml sterile Spritzen (BD). </li></ol> Schritt 2: Gewebeproben, Isolation und mechanischen Dissoziation 1-4 Die Gewebeproben werden unter einer sterilen Abzug durchgeführt und beinhalten frisch geernteten Sehnen und Muskulatur werden aus der Tibia oder soleus Muskeln der hinteren Gliedmaßen von Wildtyp-Mäusen (weiblich C57BL/6J, 4-5 Wochen alt oder jünger, Jackson erhalten Laboratory). Jede sichtbare Bindegewebe, Blutgefäße und Fettgewebe werden dann von der Biopsie-Proben entnommen. Die Gewebe der Sehnen und Muskeln werden dann vorsichtig unter einem chirurgischen Präpariermikroskop um Reste von Haut und Knochen entfernen identifiziert und in eine Schüssel mit kaltem (4 ° C) HBSS (ergänzt durch Zusatz von 5% der FBS). Die isolierten Gewebe werden dann separat auf Kollagen beschichtete Platten mit kaltem HBSS gelegt und wird zerkleinert (&lt;1x1 mm 3) in einem groben Brei mit Mikro-Schere und / oder Skalpellklingen werden. Schritt 3: Enzymatische Verdauung der Gewebe: Das zerkleinerte Gewebe wird dann enzymatisch gespalten durch eine Reihe von Schritten 2,4,5 <ol><li> Übertragen Sie die zerkleinerten Gewebes Schlämmen in einzelne 15-ml-Tuben und Zentrifuge bei ~ 3500 rpm bei 4 ° C für 5 Minuten. </li><li> Entfernen Sie den Überstand mit HBSS gewaschen und wiederholen Sie die Zentrifugation erneut. </li><li> Entfernen Sie den Überstand und verdauen diese Schlämme durch Zugabe von 10 ml vorgewärmtem 0,2% Kollagenase Typ XI (Sigma). Inkubieren für 60 Minuten bei 37 ° C unter ständigem vorsichtiges Schwenken der Röhrchen. Alternativ kann durch die Hand zu schütteln alle 10 min. </li><li> Zentrifuge und resuspendieren diese Schlämme in 10 ml Dispase (2,4 Einheiten / ml, Gibco)-Lösung. Inkubieren für 45 Minuten bei 37 ° C, Schütteln mit der Hand oder sanft schaukelnden alle 10 min. </li><li> Zentrifuge und resuspendieren diese Schlämme in 10 ml 0,2% Trypsin HBSS-Lösung. Inkubation wird basierend auf dem Ausmaß, in dem die einzelnen Zellen aus dem Gewebe, das durch die Beobachtung der Aufhängung unter dem Mikroskop durchgeführt werden können freigesetzt werden, variieren. Es ist nicht zu empfehlen, 30 Minuten bei 37 ° C überschreiten mit Umdrehen der Röhrchen oder sanft schaukelnden alle 10 min. </li><li> Centrifuge die resultierende Zelles und Resuspension des Zellpellets in 10 ml Medium Proliferation (PM). </li><li> Dissoziieren die Zellsuspension, indem der Extrakt durch eine Reihe von Nadeln. Die Zellen werden dann 2 mal durch eine 18G weitergegeben, dann 23G, und schließlich eine 27G Nadel. </li><li> Pass der Zellextrakt durch eine 70-um Zellsieb. </li></ol> Schritt 4: Preplating unterschiedliche Zellpopulationen auf die Zelladhäsion bei 1-3 basiert isolieren <ol><li> Zentrifuge und resuspendieren die Zellpellets in Proliferation Medium. </li><li> Platte der Zelle Mischungen auf eine Kollagen-beschichtete T-25-Kolben (oder 60 mm-Schale) und markieren Sie ihn als PP1. Die Zellsuspension sollte besitzt eine große Anzahl von brechenden Kernen und andere Ablagerungen unter dem Mikroskop beobachtet werden. </li><li> Bei 37 ° C in einer befeuchteten, 5% CO 2-Inkubator für 2 Stunden. </li><li> Transfer nicht anhaftenden Zellen zu einem neuen Collagen-beschichteten T-25-Kolben (oder 60 mm-Schale) und markieren Sie als PP2. 5 ml PM in die Platte gekennzeichnet PP1. Die frühen anhaftenden Zellen, die in den Kolben befestigen sind meist Myofibroblasten 3. </li><li> Bringen Sie den Kolben in den Inkubator für weitere 24 Stunden, Transfer nicht anhaftenden Zellen zu einem neuen Collagen-beschichteten T-25-Kolben (oder Gericht) und markieren Sie ihn als PP3. 5 ml Medium Proliferation in der Platte bezeichnet PP2. Diese anhaftenden Zellen, die zu dieser Kolben befestigen sind meist Fibroblasten 2,3. </li><li> Zurück Flaschen in den Inkubator und wiederholen Sie den Vorgang in einem anderen 24 Stunden, bis der Bevölkerung PP6 erstellt. Halten Sie jede Gruppe von suspendierten Zellen und lassen sie sich zu vermehren und untersuchte sie regelmäßig mit einem inversen Mikroskop. PP3-PP4 sind meist Myoblasten während Muskel-Satelliten-Zellen sind oft vor allem in PP5 2 zu sehen. </li><li> Die meisten der langsam anhaftenden Zellen in der Regel durch PP6 befestigen. In diesem Stadium erscheinen die Zellen kleine, runde, Lichtbrechung und sind sehr spärlich an Zahl und gelten als eine Stammzelle Bevölkerung 1,2 sein. </li></ol> Schritt 5: Identifizierung und Ausbau von isolierten Stammzellen <ol><li> Die isolierte PP6 Zellen sind sehr gering an Zahl und müssen in FBS reiche Proliferation Medium (20% FBS in DMEM), die täglich gewechselt beibehalten werden. Die meisten Zellen im PP6 Kultur sterben in den folgenden 1-2 Wochen der Kultivierung, sondern eine große, gesunde PP6 Bevölkerung ist in der Regel nach einer weiteren 1-2 Wochen nach der Erweiterung geschaffen. Die Stammzellen aus Mäusen isoliert hoch ausdrückliche Stammzellen-Antigen 1 (SCA1), CD34, fetale Leber-Kinase 1 (Flk1) und andere Stammzellen Marker 1,2,6,7, die visualisiert werden über immunzytochemische Färbung kann. Diese Stammzellen weisen auch mehrere Differenzierung Kapazitäten 3,6,8. </li><li> Die isolierten Zellen werden gepflegt und ausgebaut zu einem niedrigen Zelldichte in der Kultur (12-Well-Schalen bei 50-100 Zellen / Well oder &lt;20-30% Konfluenz in der Flasche oder Schale), die Differenzierung 1,2 zu vermeiden. Sehr wenige Zellen bilden Klone während der Expansion, und diese geklonten Stammzellen können lange Zeit Proliferation (LTP) für jeden anderen Kompetenz-Studien unterzogen. Das geklonte Stammzellen auch speichern kann in flüssigem Stickstoff mit allgemeinen Zelle Speicherung Verfahren, z. B. PM-Medium mit 10% DMSO. Machen Sie eine Reihe von niedrigen Durchgang Aktien der Stammzellen für alle anderen Backup-Studien. </li></ol>

<ol>
	<li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu-Petersen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+population+of+muscle+stem+cells+in+mice:+potential+for+muscle+regeneration.">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li><li>Li, Y. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle.">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li><li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &. a. m. p. ;. a. m. p., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse.">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li><li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics.">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing.">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers.">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential.">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li><li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+based+on+muscle+stem+cells.">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li><li>Gates, C. B., Karthikeyan, T., Fu, F. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells.">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li></ol>

Es wurde erkannt, dass einige adulten Geweben mehrere Stammzellen enthalten. In den letzten Jahren des Studiums, haben Stammzellen in das weiche Gewebe des Bewegungsapparates, einschließlich Skelettmuskel und Sehne festgestellt. Das aktuelle Protokoll Einzelheiten eines Verfahrens, wie die modifizierte Vorbeschichtungszyklus Technik, die bereits erfolgreich in unserem Labor verwendet werden, um adulte Stammzellen aus Sehnen und Skelettmuskulatur zu isolieren bekannt. Mit dem modifizierten Vorbeschichtungszyklus Technik...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#11995-073</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#10437-028</td>
<td>&nbsp;</td>
<tr>
<td>Heat-inactivated horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#26050-088</td>
<td>&nbsp;</td>
<tr>
<td>Chick embryo extract</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>#CE650T-10</td>
<td>&nbsp;</td>
<tr>
<td>100U ml penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15140-122</td>
<td>&nbsp;</td>
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<td>Dulbecco&#x2019;s PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#14190-250</td>
<td>&nbsp;</td>
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<td>Hank&#x2019;s Buffered Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#24020-117</td>
<td>&nbsp;</td>
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<td>Collagenase type XI 0.2%(wt/vol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C7657</td>
<td>&nbsp;</td>
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<td>Dispase2.4U ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#17105-041</td>
<td>&nbsp;</td>
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<td>Trypsin-EDTA0.5%(wt/vol) (10x;5g liter trypsin (1:250)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15400-054</td>
<td>&nbsp;</td>
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<td>Collagen for coating flask : Type I collagen derived from calf skin 0.1 g liter</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C-9791</td>
<td>&nbsp;</td>
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<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>#D-2650</td>
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isolating stem cells from soft musculoskeletal tissues

Adulte Stammzellen sind seit langem in Bezug auf ihre Anwendung in der regenerativen Medizin diskutiert. Adulte Stammzellen haben eine große Aufregung für die Behandlung von verletzten und erkrankten Geweben durch ihre beeindruckenden Fähigkeiten, um multi-lineage Zelldifferenzierung und ihre Selbst-Erneuerung unterzogen Fähigkeit generiert. Am wichtigsten ist, haben diese Eigenschaften machten sie vorteilhaft für die Verwendung bei der autologen Stammzelltransplantation Therapien. Das aktuelle Protokoll wird der Leser auf die veränderten Vorbeschichtungszyklus Technik, bei der Weichteile des Bewegungsapparates, zB Sehnen und Muskeln, sind 1 einführen<sup&gt; St</sup&gt; Enzymatisch gespalten und dann in Kollagen beschichtet Kolben mit Medium. Der Überstand, der mittleren und die restlichen schwimmenden Zellen zusammengesetzt ist, ist seriell übertragen täglich zu neuen Flaschen. Die Stammzellen sind die langsamsten auf die Kolben, welche dauert in der Regel beträgt 5-7 Tage (serielle Übertragungen oder preplates) einzuhalten. Durch den Einsatz dieser Technik, präsentieren adulten Stammzellen in diesen Geweben kann leicht durch relativ nicht-invasive Verfahren gewonnen werden.

Watch this Scientific Journal Video about Isolating Stem Cells from Soft Musculoskeletal Tissues at JoVE.com

Isolating Stem Cells from Soft Musculoskeletal Tissues

Isolating adulten Stammzellen aus Muskel-Skelett Weichteile auf der Grundlage der Zelle Einhaltung Geschwindigkeit Kolben.

Isolating Stammzellen aus Soft-Gewebe des Bewegungsapparates

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

isolating-stem-cells-from-soft-musculoskeletal-tissues

Research

JoVE Journal

Biology

13.3K Aufrufe.  Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC. Isolating adulten Stammzellen aus Muskel-Skelett Weichteile auf der Grundlage der Zelle Einhaltung Geschwindigkeit Kolben.

Serie: Isolating Stem Cells from Soft Musculoskeletal Tissues

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

Ableitung von hämatopoetischen Stammzellen aus embryonalen Stammzellen der Maus

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

Forschung

Biologie

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Chromatin Immunopräzipitation von humanen embryonalen Stammzellen

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Ableitung der Maus Trophoblast Stammzellen aus Blastozysten

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

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolierung von Stammzellen aus menschlichen Bauchspeicheldrüsenkrebs Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Selektieren und Isolieren Colonies of Human induzierten pluripotenten Stammzellen aus adulten Fibroblasten Umprogrammierte

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Isolieren Primäre Melanozyten-ähnlichen Zellen aus der Maus Herz

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

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 ...

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells&#8217; (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

Isolieren von Stammzellen des Darms von Adult<em&gt; Drosophila</em&gt; Mitteldärmen durch FACS, um Stammzellverhalten Studie während des Alterns

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly ...