Die Autoren danken Janet Rossant für das ursprüngliche Protokoll. Diese Studie wurde vom National Institute of Health gewähren CA106308 zu WH unterstützt.

In diesem Protokoll, beschreiben wir die Herstellung von Maus-Blastozysten und die Einrichtung von TS-Zelllinien. Allgemeine Maus Manipulationen vor der Sammlung von Blastozysten, einschließlich der Einrichtung für die natürliche Paarung und die Induktion von Super Eisprung folgen grundsätzlich dem Standard-Protokoll von Nagy et al dargestellt. 2003 (pp146-150). Ableitung und Wartung von TS-Zellen <ol><li> Setup-Zucht Kreuzung zwischen Mäusen von Interesse. </li><li> Bereiten embryonalen Maus-Fibroblasten (MEFs) als Feeder-Zellen. Zwei Tage vor der Sammlung von Blastozysten, MEFs (2 x 10 6 Zellen) werden in 100 mm-Schalen plattiert. Am nächsten Tag sind diese Zellen mit 10 ml TS-Medium mit Mitomycin C (20 mg / ml) für ein 2-Stunden-Inkubation bei 37 ° C, 5% CO 2 behandelt. Dies wird durch Waschen der Zellen zweimal mit PBS folgten. Die Mitomycin C behandelten Zellen werden dann in 12-Well-Platten (1 x 10 5 Zellen / well) ausgesät. </li><li> Am Tag der Blastozyste Sammlung (3,5 dpc, die so genannte Tag), ist MEF Kulturmedium durch TS-Medium plus 1x FGF4/Heparin (0,5 ml / well) ersetzt. </li><li> Vorbereitung für die Sammlung der Gebärmutter: euthanize der Maus, und suchen die Gebärmutter (Abbildung 1A). </li><li> Entfernen der Gebärmutter, indem Sie es mit einer Pinzette an der Zervix (befindet sich hinter der Blase) und quer durch die Kreuzung der Gebärmutter und des Gebärmutterhalses (Abbildung 1B). Ziehen Sie die Gebärmutter, schneiden Sie die Membran-und Fettgewebe weggeschnitten und der Gebärmutter unter der Kreuzung mit der Eileiter (Abbildung 1C). Legen Sie die uteri (Abb. 1D) in einem kleinen Volumen (0,2 ml) von TS-Medium in einem 60 mm Platte. </li><li> Flush jedes Uterushorn mit 1 ml TS-Medium unter Verwendung einer 1-ml-Spritze mit einer 26-Gauge-Nadel. Führen Sie die Nadel in die Basis des Uterus und Flush langsam in eine neue 60 mm-Platte mit 1 ml TS Medium. Die Gebärmutter ist aufgeblasen und voll beim Spülen verlängert. </li><li> Sammeln und Transfer der Blastozysten vorsichtig in einem sauberen 12-Well-Platte mit TS-Medium mit einer Mund-kontrollierten Pipette. Wash Blastozysten dreimal durch serielle Übertragung durch TS Medium in 12-Well-Platten. Legen Sie eine Blastozyste pro Well (Abbildung 2A) in einer 12-Well-Platte mit der Mitomycin C-behandelten MEF Feeder und Kultur bei 37 ° C, 5% CO 2. </li><li> Die Blastozysten werden von zona pellucida schlüpfen und heften sich an den Brunnen in 24 bis 36 Stunden. Ein kleiner Auswuchs ohne weiteres an Tag 3 beobachtet werden. Feed the Kulturen mit 500 ul frisch TS-Medium plus 1x FGF4/Heparin. </li><li> Am Tag 4 oder 5 sollte die Blastozyste Auswuchs bereit sein, für die Aufschlüsselung in Abhängigkeit von ihrer Größe. Die ideale Größe für TS Zelle Disaggregation ist in Abbildung 3B dargestellt. Waschen Sie die Kulturen einmal mit 0,5 ml PBS. Add 100 ml 0,25% Trypsin / EDTA und Inkubation für 5 Minuten bei 37 ° C, 5% CO 2. Break the Auswuchs in kleine Klumpen von Auf-und Abpipettieren kräftig mit einem P100 pipetteman. Stoppen Sie die Trypsinierung indem TS-70cm-1.5F4H (30% TS-Medium, 70% MEF-CM plus 1.5x FGF4/Heparin) in den Brunnen. Ändern Medium 8 Stunden nach der Disaggregation und weiterhin Zellen ernähren alle 2 Tage. </li><li> Zwischen Tag 6 und 10 (sehr variabel) TS Zellkolonien beobachtet werden können. Sie haben flache, epithelialen Blatt Morphologie mit einer klaren Kolonie Grenze (Abbildung 2C). Weiter zu den Kulturen Feed alle 2 Tage </li><li> Wenn TS-Zellen 50% Konfluenz (in der Regel zwischen den Tagen 15 und 20) zu erreichen, einmal waschen mit PBS und 100 ml 0,25% Trypsin / EDTA. Pipette auf und ab während Trypsinierung zu einem in der Nähe Einzel-Zell-Suspension zu gewährleisten. Stoppen Sie die Trypsinierung indem TS-70cm-1.5F4H und Übertragung auf neue 6-well-oder 60mm-Platten mit der Mitomycin C-behandelten MEF Feeder (2,5 x 10 5 cells/6-well oder 5 x10 5 cells/60mm Platte). </li><li> Ändern Medium 8 Stunden nach der Passage und auch weiterhin die Kulturen Feed alle 2 Tage. </li><li> Nach ein oder zwei weitere Passagen (3-5 Tage zwischen den Kanälen) auf der MEF Speiser, kann TS-Zellen ohne sie kultiviert werden. Da MEFs die Kultur Platte viel schneller als TS-Zellen nach Trypsinierung legen, können solche Unterschiede in der Rate Anhänger genutzt werden, um TS-Zellen aus dem MEF Anleger trennen. Nach dem Passieren der kultivierten Zellen, brüten sie bei 37 ° C, 5% CO 2 für 30 min. Die meisten MEFs wird auf die Platte legen und die TS Bevölkerung bleibt in der Schwebe. Übertragen Sie den Überstand auf eine neue Platte. Wiederholen Sie diesen Vorgang für mehrere Gänge zu einem reinen TS Bevölkerung zu erhalten. </li><li> Pflegen Sie TS-Zellen in TS-70cm-1.5F4H und geben sie alle 4 Tage (von 1.10 bis 01.20 split) oder wenn die Kultur erreicht 70% Konfluenz. Vorbei an den Zellen mit hoher Dichte (höher als 1:5) oder kultiviert werden, wenn übermäßig konfluenten ihre Differenzierung führen kann. </li><li> Die Identität des TS-Zellen können durch Immunfärbung eines Zell-Marker, Cdx2 (Abbildung 3) bestätigt werden. </li></ol> Freezing TS-Zellen <ol><li> Bereiten Sie 2x Einfriermedium (50% FBS, 30% TS mittleren und 20% DMSO). </li><li> Collect TS-Zellen durch Trypsinierung, durch Zentrifugation bei 450g für 6 min folgte. Resuspendieren sie in TS-Medium mit gleichen Volumen 2x Einfriermedium. </li><li> Einfrieren der Zellen langsam mit einem Isopropylalkohol Kammer bei -70 ° C und Transfer zum flüssigen Stickstoff nach 24-48 Stunden. </li><li> A 60 mm-Platte (70% konfluent) ist in der Regel in 2 Fläschchen aufbewahrt. </li></ol> Auftauen TS-Zellen <ol><li> Schnell Tauwetter das Fläschchen in 37 ° C, sammeln sie in 10 ml TS Medien durch Zentrifugation (450g, 6 min) und Resuspension in 30% TS Medien, 70cm-1.5F4H. </li><li> Ein Fläschchen von Zellen in einer 60 mm-Platte ausgesät. </li></ol> TS-Zell-Differenzierung in TGC Die Differenzierung der TS-Zellen in Trophoblast Riesenzellen (TGCs) kann durch den Abzug der MEF-CM, FGF4 und Heparin induziert werden. Das TGC-Phänotyp (große Kerne) kann vier Tage nach der Induktion nachgewiesen werden (Tanaka et al., 1998 und Chiu et al. 2008). Immunfärbung von p450scc erkennen die Expression dieses TGC Marker in der differenzierten Kultur (Abbildung 4A). Durchflusszytometrische Analyse der PI (Propidiumiodid) gefärbten Zellen zeigt weiter das Vorhandensein von differenzierten Zellen mit höheren DNA-Gehalt (Abbildung 4B). Diese Ergebnisse legen nahe, dass TS-Zellen Endoreduplikation unterziehen, um polyploiden TGCs geworden. Repräsentative Ergebnisse <img src="/files/ftp_upload/1964/1964fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Dissection der Maus uteri. (A) Lage der Gebärmutter ist durch Pfeile angedeutet. (B) über die Kreuzung der Gebärmutter und des Gebärmutterhalses Cut, wie dargestellt. (C) Cut der Gebärmutter direkt unterhalb der Eileiter (OD) unter dem Fruchtknoten (OV). (D) Bild des Uterus. <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Representative Bilder illustrieren Maus Blastozyste, Blastozyste Auswuchs und Trophoblast Kolonie. (A) Eine Maus Blastozyste an E3.5 gesammelt wird angezeigt. ICM, innere Zellmasse, MT, Wandmalerei Trophektoderm; PT, polar Trophektoderm. (B) A Trophoblasten Auswuchs am MEF Feeder wird angezeigt. TS, Trophoblast Stammzelle. (C) A Trophoblasten Stammzellen Kolonie zeigt einen klaren Vorsprung am MEF Feeder. Die Pfeile zeigen die Zellgrenze. <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Identifizierung von TS-Zellen durch Immunfärbung Analyse. TS (AD) und ES (Steuerung, EH)-Zellen unterliegen Marker-Identifizierung. Die Zellen werden mit Antikörpern gegen Oct4 (A, E) oder Cdx2 (B, F) zu erkennen immungefärbt und gegengefärbt von DAPI (C, G) in fusionierten Bildern dargestellt (D, H). Scale-bar, 50 pm. <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Differenzierung der TS-Zellen in vitro. (A) Undifferenzierte (Undiff) oder differenziert (Diff)-Zellen in Kultur für den Ausdruck einer TGC-Marker untersucht, gegengefärbt p450scc (grün), mit DAPI (rot). (B) Durchflusszytometrische Analyse der differenzierten Zellen mit PI gefärbt, misst den DNA-Gehalt (M1, zwei Minuten vor vier Exemplare; M2, mehr als vier Exemplare). Die M2 Bevölkerung stellt die polyploiden Trophoblastzellen.

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

In diesem Video zeigen wir, den Prozess zu E3.5 Blastozysten aus uteri und experimentellen Verfahren zu sammeln, um TS-Zelllinien zu etablieren. Wir beschreiben auch die Voraussetzung für die Stammzell-Seins der TS-Zellen zu erhalten und deren Differenzierung in differenzierten Zellen zu induzieren. Zwei entscheidende Schritte zur reinen TS-Zelllinien zu erhalten sind das Timing für Auswuchs Disaggregation (Schritt 9) und die Verarbeitung der ersten Passage (Schritt 11). Es wurde berichtet, dass primitive Endoderm-abg...

Reagenzien TS-Medium: RPMI-1640, 20% FBS, 1 mM Natriumpyruvat, 100 mM beta-Mercaptoethanol und 100 mg / ml Penicillin-Streptomycin. Mitomycin C: Lösen Sie 2 mg Mitomycin C (Sigma M-0503) in 2 ml PBS und stell &#39;das 2 ml Mischung in 200 ml TS-Medium (10 ng / ml). Machen Sie 20 Portionen (10 ml) und bei -20 ° C bis zur Verwendung. Fügen Sie ein Aliquot pro 100 mm-Platte. Maus embryonale Fibroblasten-Zustand Medium (MEF-CM): Platte MEFs in 100 mm-Platten (1 x 10 6 / Platte). Am nächsten Tag, 10 ml TS Medium pro Schale und weiterhin Kultur für 48 Stunden. Sammeln Sie das Kulturmedium, filtern (0,45 mm) und bei -20 ° C in 35 ml Aliquots. Thaw jedes Aliquot, wenn nötig, die bei 4 gespeichert werden können ° C. Die MEFs kann für zwei weitere Chargen von CM vorbereiten, bevor sie konfluent geworden sein. PBS / BSA: Lösen BSA, Fraktion V (Sigma A3311, 0,1% (w / v)) in PBS (10 ml), filtriert durch ein 0,45 mm Spritzenfilter und machen 1 ml Aliquots in 1,5 ml Röhrchen. Lagerung bei -70 ° C und Auftauen einer Röhre, wenn benötigt, um FGF4 vorzubereiten. FGF4 (1000x): Resuspendieren lyophilisiert FGF4 (Sigma F8424, 25 mg) mit 1 ml der PBS / BSA-Lösung. Gut mischen und machen 10 Aliquots (100 ml) in 1,5 ml Röhrchen und lagern bei -70 ° C. Thaw jedes Röhrchen bei Bedarf und speichern Sie die restlichen bei 4 ° C, nicht wieder einfrieren. Verdünnen 1000 mal in TS-Medium auf 1x FGF4 (25 ng / ml) zu erhalten. Heparin (1000x): Resuspendieren Heparin (Sigma H3149, 10.000 Einheiten) in PBS bis zu einer Endkonzentration von 1 mg / ml (1000x). Machen Sie 100 ml Aliquots in 1,5 ml Röhrchen und lagern bei -70 ° C. Thaw Aliquots bei Bedarf und speichern Sie die restlichen bei 4 ° C, nicht wieder einfrieren. Verdünnen 1000 mal in TS-Medium auf 1x Heparin (1 ng / ml) zu erhalten.

derivation of mouse trophoblast stem cells from blastocysts

Spezifikation der Trophektoderm ist eine der frühesten Differenzierung Veranstaltungen von Säugetier-Entwicklung. Der Trophoblast Linie aus dem Trophektoderm vermittelt Implantation abgeleitet und generiert die fetalen Teil der Plazenta. Als Ergebnis ist die Entwicklung dieser Linie wichtig für den Embryo Überleben. Herleitung der Trophoblasten Stammzellen (TS)-Zellen von Maus Blastozysten wurde zuerst von Tanaka beschrieben<em&gt; Et al.</em&gt; 1998. Die Fähigkeit der TS-Zellen, die Trophoblasten spezifische Eigenschaft und ihre Expression von Bühnen-und Zelltyp-spezifische Marker nach entsprechender Stimulation erhalten eine wertvolle Modellsystem, um die Trophoblasten Abstammung Entwicklung zu untersuchen, wobei Rekapitulation frühen Plazentation Veranstaltungen. Darüber hinaus sind Trophoblastzellen einer der wenigen somatische Zelltypen unterziehen natürliche Genome Amplification. Obwohl die zugrunde liegenden molekularen Mechanismen Trophoblasten Polyploidisierung begonnen haben, zu entwirren, bleibt die physiologische Rolle und der Vorteil der Trophoblast Genomamplifikationstechnik weitgehend ungeklärt. Die Entwicklung der diploiden Stammzellen in polyploiden Trophoblast-Zellen in Kultur macht diese<em&gt; Ex vivo</em&gt; System das perfekte Werkzeug für die Aufklärung der regulatorischen Mechanismus der Genom-Replikation und Instabilität in Gesundheit und Krankheit. Hier beschreiben wir ein Protokoll über die früheren Berichte mit Modifikationen in Chiu veröffentlichten<em&gt; Et al.</em&gt; 2008.

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

In diesem Video zeigen wir die Isolierung der Maus Blastozysten und die Ableitung der Trophoblast-Stammzellen aus Blastozysten. Wir beschreiben auch Bedingungen für die Wartung der Stammzell-Eigenschaft sowie die Induktion der Differenzierung in Kultur.

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 ...

derivation-of-mouse-trophoblast-stem-cells-from-blastocysts

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JoVE Journal

Biology

18.1K Aufrufe.  University of Rochester. In diesem Video zeigen wir die Isolierung der Maus Blastozysten und die Ableitung der Trophoblast-Stammzellen aus Blastozysten. Wir beschreiben auch Bedingungen für die Wartung der Stammzell-Eigenschaft sowie die Induktion der Differenzierung in Kultur.

Serie: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

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

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Gewinnung menschlicher embryonaler Stammzellen durch Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

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 of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Isolation of Primary Maus Trophoblastzellen und Trophoblastinvasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Serielle Anreicherung von Spermatogonien Stamm-und Progenitorzellen (SSC) in der Kultur für die Ableitung des langfristigen adulten Maus SSC Linien

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

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 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 ...