Gli autori ringraziano Janet Rossant per il protocollo originale. Questo studio è stato sostenuto dal National Institute of Health concedere CA106308 a WH.

In questo protocollo, si descrive la preparazione di blastocisti mouse e la creazione di linee cellulari TS. Manipolazioni del mouse generale prima della raccolta di blastocisti, tra cui il set-up per la monta naturale e l&#39;induzione dell&#39;ovulazione super, in fondo, il protocollo standard illustrato da Nagy et al. 2003 (pp146-150). Derivazione e il mantenimento delle cellule TS <ol><li> Setup croce riproduzione tra i topi di interesse. </li><li> Preparare fibroblasti embrionali di topo (MEF) come cellule nutrici. Due giorni prima della raccolta delle blastocisti, MEF (2 x 10 6 cellule) sono placcati in 100 piatti mm. Il giorno dopo, queste cellule vengono trattate con 10 ml di terreno TS contenente mitomicina C (20 mg / ml) per un 2 ore di incubazione a 37 ° C, 5% di CO 2. Questo è seguito da lavaggio delle cellule due volte con PBS. Le cellule C mitomicina trattate vengono poi seminate a 12-pozzetti (1 x 10 5 cellule / pozzetto). </li><li> Il giorno della raccolta blastocisti (3,5 DPC, indicato come primo giorno), terreno di coltura è sostituito dal MEF TS medio più 1x FGF4/Heparin (0,5 ml / pozzetto). </li><li> Preparazione per la raccolta dell &#39;utero: eutanasia con il mouse e individuare l&#39;utero (Figura 1A). </li><li> Togliere l&#39;utero afferrandolo con una pinza al collo dell&#39;utero (che si trova dietro la vescica) e tagliare attraverso la giunzione dell&#39;utero e della cervice uterina (Figura 1B). Tirare l&#39;utero, tagliare la membrana e nei tessuti grassi e tagliare via l&#39;utero sotto l&#39;incrocio con la ovidotto (Figura 1C). Luogo cervice (Figura 1D) in un piccolo volume (0,2 ml) di media TS in un piatto di 60 mm. </li><li> Invia ogni corno dell&#39;utero con 1 ml di terreno TS utilizzando una siringa da 1 ml con una 26-gauge. Inserire l&#39;ago nella base di utero e sciacquare lentamente in un nuovo 60 mm con 1 media TS ml. L&#39;utero deve essere gonfiato ed esteso completamente durante il lavaggio. </li><li> Raccogliere e trasferire la blastocisti con cura in un ambiente pulito 12 pozzetti contenenti medio TS con una bocca controllato pipetta. Lavare blastocisti tre volte trasferimento seriale attraverso media TS a 12-pozzetti. Mettere una blastocisti per bene (Figura 2A) in un 12-pozzetti contenenti la mitomicina C trattati con alimentatori MEF e della cultura a 37 ° C, 5% di CO 2. </li><li> La blastocisti si schiuderanno dalla zona pellucida e allegare ai pozzetti in 24 a 36 ore. Una piccola escrescenza sarà prontamente osservato il giorno 3. Alimentare le culture con 500 microlitri TS medio fresco più 1x FGF4/Heparin. </li><li> Il giorno 4 o 5, la conseguenza blastocisti dovrebbe essere pronto per la disaggregazione a seconda della sua grandezza. La dimensione ideale per la disaggregazione delle cellule TS è illustrato in Figura 3B. Lavare le culture una volta con 0,5 ml di PBS. Aggiungere 100 ml 0,25% tripsina / EDTA e incubare per 5 minuti a 37 ° C, 5% di CO 2. Rompere il escrescenza in piccoli grumi pipettando su e giù vigorosamente con un pipetteman P100. Fermare il tripsinizzazione aggiungendo TS-70cm-1.5F4H (medio TS 30%, 70% MEF-CM più FGF4/Heparin 1.5x) nel pozzo. Cambia media 8 ore dopo la disaggregazione e continuare ad alimentare le cellule ogni 2 giorni. </li><li> Tra i giorni 6 e 10 (molto variabile) colonie di cellule TS può essere osservato. Hanno una lastra piana e come morfologia epiteliale con un confine chiaro colonia (Figura 2C). Continuano ad alimentare le culture ogni 2 giorni </li><li> Quando le cellule TS raggiungere il 50% confluenza (di solito tra i giorni 15 e 20), lavare una volta con PBS e aggiungere 100 ml 0,25% tripsina / EDTA. Pipetta su e giù durante tripsinizzazione per garantire un vicino cella singola sospensione. Fermare il tripsinizzazione aggiungendo TS-70cm-1.5F4H e trasferimento in nuovi piatti da 6 o 60 millimetri e contiene il mitomicina C trattati con alimentatori MEF (2,5 x 10 5 cells/6-well o 5 x10 5 cells/60mm piastra). </li><li> Cambia media 8 ore dopo il passaggio e continuare ad alimentare le culture ogni 2 giorni. </li><li> Dopo uno o due in più passaggi (3-5 giorni tra i passaggi) sul alimentatori MEF, le cellule possono essere coltivate TS senza di loro. Perché MEF allegare alla piastra di coltura molto più veloce rispetto alle cellule TS dopo tripsinizzazione, tale differenza nel tasso aderente può essere utilizzata per separare le cellule TS dal alimentatori MEF. Dopo aver superato le cellule in coltura, li incubare a 37 ° C, 5% di CO 2 per 30 min. La maggior parte MEF attribuirà alla piastra e la popolazione TS rimane in sospensione. Trasferire il supernatante in una nuova piastra. Ripetere questo processo per vari passaggi per ottenere una popolazione pura TS. </li><li> Mantenere le cellule TS a TS-70cm-1.5F4H e passarli ogni 4 giorni (1:10-1:20 split) o ​​quando la cultura raggiunge il 70% confluenza. Passando le cellule con alta densità (superiore a dividere 1:5) o coltura quando eccessivamente confluenti può causare la loro differenziazione. </li><li> L&#39;identità delle cellule TS può essere confermata mediante immunocolorazione di un marker delle cellule, Cdx2 (Figura 3). </li></ol> Celle di congelamento TS <ol><li> Preparare 2x medio congelamento (50% FBS, 30 di media TS% e 20% DMSO). </li><li> Cocellule TS llect da tripsinizzazione, seguita da centrifugazione a 450g per 6 min. Risospendere in media TS con volume equivalente di mezzo di congelamento 2x. </li><li> Congelare le cellule lentamente utilizzando una camera di alcool isopropilico a -70 ° C ed il trasferimento di azoto liquido, dopo 24-48 ore. </li><li> Un piatto 60 mm (70% confluenti) è di solito tenuti in 2 flaconi. </li></ol> Scongelamento cellule TS <ol><li> Scongelare rapidamente il flacone a 37 ° C, raccoglierli in 10 ml mezzi TS mediante centrifugazione (450g, 6 min), e risospendere in 30% TS media, 70cm-1.5F4H. </li><li> Un flaconcino di cellule seminate in una piastra 60 mm. </li></ol> TS differenziazione cellulare in TGC Differenziazione delle cellule TS in cellule giganti trofoblasto (TGCs) può essere indotta dal ritiro del MEF-CM, FGF4 ed eparina. Il fenotipo TGC (nuclei di grandi dimensioni) possono essere individuati quattro giorni dopo l&#39;induzione (Tanaka et al., 1998 e Chiu et al. 2008). Immunocolorazione p450scc di rilevare l&#39;espressione di questo marcatore TGC nella cultura differenziata (Figura 4A). Citometria a flusso di PI (ioduro di propidio) cellule colorate dimostra ulteriormente la presenza di cellule differenziate con contenuto di DNA superiore (Figura 4B). Questi risultati suggeriscono che le cellule subiscono TS endoreduplicazione a diventare TGCs poliploidi. Rappresentante Risultati <img src="/files/ftp_upload/1964/1964fig1.jpg" alt="Figura 1" /> Figura 1. Dissezione di uteri mouse. (A) Luogo della cervice è indicata dalle frecce. (B) Tagliare attraverso la giunzione dell&#39;utero e della cervice uterina, come mostrato. (C) Tagliare l&#39;utero appena sotto la ovidotto (OD) sotto l&#39;ovaio (OV). (D) Immagine della cervice. <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="Figura 2" /> Immagini figura rappresentativa 2. Illustrare blastocisti del mouse, escrescenza blastocisti e colonia trofoblasto. (A) Un topo blastocisti raccolti E3.5 viene mostrato. ICM, la massa cellulare interna, MT, trophectoderm murale, PT, trophectoderm polare. (B) Un escrescenza trofoblasto su linee MEF è mostrato. TS, di cellule staminali trofoblasto. (C) Una colonia trofoblasto cellule staminali mostra un chiaro vantaggio su linee MEF. Le frecce indicano il confine della cella. <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="Figura 3" /> Figura 3. Identificazione di cellule TS immunocolorazione analisi. TS (AD) ed ES (controllo, EH) le cellule sono sottoposte a identificazione marcatore. Le cellule sono immunoistochimica con anticorpi per riconoscere Oct4 (A, E) o Cdx2 (B, F), e di contrasto con DAPI (C, G) ha presentato nelle immagini fuse (D, H). Scala bar, 50 micron. <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="Figura 4" /> Figura 4. Differenziazione delle cellule TS in vitro. (A) Indifferenziato (Undiff) o differenziato (Diff), le cellule in coltura sono stati esaminati per l&#39;espressione di un marker TGC, p450scc (verde), di contrasto con DAPI (rosso). (B) L&#39;analisi citofluorimetrica delle cellule differenziate, colorate con PI, misura il contenuto del DNA (M1, da due a quattro copie, M2, più di quattro copie). La popolazione M2 rappresenta il trofoblasto cellule poliploidi.

<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>

Nessun conflitto di interessi dichiarati.

In questo video, dimostriamo il processo per la raccolta E3.5 blastocisti dalle procedure di uteri e sperimentali per stabilire linee di cellule TS. Abbiamo anche descrivere la condizione per mantenere la staminalità delle cellule TS e per indurre la loro differenziazione in cellule differenziate. A due passi fondamentali per ottenere linee cellulari puro TS sono i tempi per la disaggregazione escrescenza (punto 9) e la lavorazione del primo passaggio (passo 11). E &#39;stato riportato che endoderma primitivo cellule d...

Reagenti TS medio: RPMI-1640, FBS 20%, 1 piruvato di sodio mm, 100 mm beta-mercaptoetanolo e 100 mg / ml di penicillina-streptomicina. Mitomicina C: Sciogliere 2 mg di mitomicina C (Sigma M-0503) in 2 ml di PBS e aggiungere questa miscela 2 ml in 200 ml di mezzo di TS (10 ng / ml). Fare 20 aliquote (10 ml) e conservare a -20 ° C fino al momento dell&#39;uso. Aggiungere una aliquota per 100 piastra mm. Fibroblasti embrionali di topo-condizione medio (MEF-CM): Piastra MEF in 100 piastre mm (1 x 10 6 / piastra). Il giorno dopo, aggiungere 10 ml di medie TS per ogni piatto e continuare con la cultura per 48 ore. Raccogliere il terreno di coltura, filtrarli (0,45 mm) e conservare a -20 ° C in 35 aliquote da ml. Disgelo ciascuna aliquota quando necessario, che può essere conservato a 4 ° C. Il MEF può essere utilizzato per preparare due lotti più di CM prima che diventino confluenti. PBS / BSA: Sciogliere BSA, frazione V (Sigma A3311, 0,1% (w / v)) in PBS (10 ml), filtrare attraverso una siringa da 0,45 millimetri filtro e fare 1 aliquote ml in provette da 1,5 ml. Conservare a -70 ° C e scongelare un tubo quando necessario per prepararsi FGF4. FGF4 (1000x): Risospendere liofilizzato FGF4 (Sigma F8424, 25 mg) con 1 ml di PBS / BSA soluzione. Mescolare bene e fare 10 aliquote (100 ml) in provette da 1,5 ml e conservare a -70 ° C. Disgelo ogni tubo quando necessario e conservare le rimanenti a 4 ° C, non ricongelare. Diluire 1000 volte in media TS di ottenere 1x FGF4 (25 ng / ml). Eparina (1000x): Risospendere eparina (Sigma H3149, 10.000 unità) in PBS alla concentrazione finale di 1 mg / ml (1000x). Fai aliquote di 100 ml in provette da 1,5 ml e conservare a -70 ° C. Aliquote disgelo quando necessario e conservare le rimanenti a 4 ° C, non ricongelare. Diluire 1000 TS tempo medio per ottenere 1x eparina (1 ng / ml).

derivation of mouse trophoblast stem cells from blastocysts

Specificazione del trophectoderm è uno degli eventi più antichi differenziazione di sviluppo dei mammiferi. Il lignaggio trofoblasto deriva dalla impianto trophectoderm media e genera la parte fetale della placenta. Di conseguenza, lo sviluppo di questo lignaggio è essenziale per la sopravvivenza dell&#39;embrione. Derivazione di staminali trofoblasto (TS), le cellule da blastocisti mouse è stato descritto per primo da Tanaka<em&gt; Et al.</em&gt; 1998. La capacità delle cellule TS per preservare la proprietà specifica trofoblasto e la loro espressione di stage e cellule di tipo-specifici marcatori dopo la stimolazione adeguata fornisce un sistema valido modello per studiare lo sviluppo lignaggio trofoblasto cui ricapitolando eventi placentazione presto. Inoltre, le cellule trofoblasto sono uno dei pochi tipi di cellule somatiche in fase di amplificazione del genoma naturale. Anche se le vie molecolari alla base poliploidizzazione trofoblasto hanno cominciato a svelare, il ruolo fisiologico e il vantaggio di amplificazione del genoma trofoblasto rimane in gran parte sfuggente. Lo sviluppo delle cellule staminali in cellule diploidi trofoblasto poliploidi in cultura fa di questo<em&gt; Ex vivo</emSistema di&gt; un ottimo strumento per chiarire il meccanismo di regolazione della replicazione del genoma e l&#39;instabilità nella salute e nella malattia. Qui si descrive un protocollo basato su precedenti rapporti con modificazioni pubblicato nel Chiu<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 questo video, dimostriamo l&#39;isolamento di blastocisti mouse e la derivazione di cellule staminali trofoblasto da blastocisti. Abbiamo anche descrivere le condizioni per il mantenimento della proprietà delle cellule staminali e l&#39;induzione della differenziazione nella cultura.

Derivazione di cellule staminali trofoblasto mouse da blastocisti

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

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18.1K Views.  University of Rochester. In questo video, dimostriamo l'isolamento di blastocisti mouse e la derivazione di cellule staminali trofoblasto da blastocisti. Abbiamo anche descrivere le condizioni per il mantenimento della proprietà delle cellule staminali e l'induzione della differenziazione nella cultura.

Video: 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.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

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 ...

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Biologia

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.

Derivazione di cellule staminali embrionali umane da 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.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

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.

Isolamento di cellule staminali della retina dalla Eye mouse

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.

Isolamento di cellule primarie trofoblasto mouse e Invasion Assay trofoblasto

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.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

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.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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.

Arricchimento di serie staminali spermatogoni e cellule progenitrici (SSC) in Cultura per la derivazione di lungo termine Lines topo adulto SSC

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.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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.

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...