Différenciation des cellules souches embryonnaires <ol><li> Recueillir un flacon à proximité de confluence des cellules souches embryonnaires (CSE), via un traitement avec la trypsine ESC. </li><li> Resuspendre les cellules dans 5 ml de milieu de différenciation et de transférer à un T25 sans gélatine couché. Incuber à 37 ° C pendant 45 minutes. Récupérer le surnageant et recueillir les cellules par centrifugation. Note: les fibroblastes embryonnaires de souris (MEF) de l&#39;ESC cultures attachent facilement à la T25 sans gélatine couché et ne sont donc appauvri de la culture au cours de cette étape de placage. Si vous n&#39;êtes pas la culture de votre ESC sur FAE, vous pouvez sauter la plaque pré-. </li><li> Resuspendre MEF appauvri CES dans les médias différenciation au 333 333 cells/50 ml. Cette concentration se traduit par 100 ml ESC/15. </li><li> L&#39;utilisation multi-canal pipette, plaque ESC à 100 ml cells/15 déposer le 15 plaques de Pétri cm 2. Avec une pipette à 8 canaux, vous devriez être en mesure d&#39;adapter environ 18-22 rangées de gouttes par plaque (environ 5 ml par plaque). Remarque: Pour ce protocole, 2-5 15 cm 2 plats de gouttes sera plus que suffisante et devrait donner 2-5 x 10 6 six jours EB cellules dérivées. </li><li> Doucement retourner les plaques d&#39;inverser gouttes. Incuber à 37 ° C CO 2 5% pendant 48 heures. </li><li> Piscine gouttes en remuant doucement les plaques et le transfert de 15 ml tube conique. Rincer les plaques avec 4-6 ml de PBS et ajouter à la même conique 15 ml. Note: Vous pouvez piscine jusqu&#39;à cinq plaques goutte suspendue. L&#39;EBS va croître car ils continuent de faire la différence et si elles sont trop concentrés, ils ont tendance à former de grosses touffes. </li><li> Autoriser commun EBS pour régler par la gravité (environ 10 minutes). Aspirer le surnageant sans déranger EBS. Doucement remettre en suspension dans 10 ml de différenciation des médias et de transfert à 10 cm de non-adhérentes boîte de Pétri. </li><li> Placer la plaque sur une plaque de Pétri agitateur à 50 tpm et incuber à 37 ° C à 5% de CO 2. </li><li> 24 heures plus tard (quatre jours de différenciation), les plaques de turbulence se concentrer EBS dans le centre du plat de Pétri. Soigneusement d&#39;échange de 50% des médias (5 ml) avec 5 ml médias Différenciation frais. Retour plateau de l&#39;incubateur pour deux jours de plus. </li></ol> EB dissociation et l&#39;infection par MSCV-HOXB4-IRES-GFP <ol><li> Sur six jours de différenciation, le transfert EBS pour tube de 15 ml conique. Laisser décanter par gravité. </li><li> Aspirer les médias et les remettre en suspension dans 10 ml de PBS. Autoriser EBS pour réinstaller par la gravité. Aspirer le PBS. </li><li> Ajouter 250 ml Mélanger l&#39;enzyme de dissociation et de 1 ml de PBS. Incuber à 37 ° C l&#39;eau de bain pendant 20 minutes, parfois tourbillonnante du tube pour mélanger des enzymes et EBS. </li><li> Ajouter 8 ml sans enzyme tampon de dissociation. </li><li> Triturer le mélange avec 5 ml pipette jusqu&#39;au EBS sont totalement dissociés (environ 10 fois; pipetage excessive augmentera la mort au sein de l&#39;EB-dérivée préparation cellulaire). <ol><li> Mélange deviendra nuageux avec cellules comme EB dissocier. </li><li> Placer la pointe de pipette contre le fond du tube conique lors de la trituration va créer une force de cisaillement qui facilitera grandement la dissociation. </li></ol></li><li> Recueillir les cellules par centrifugation. </li><li> Resuspendre EB cellules dérivées dans 5 ml de IMDM 10% et comptent utiliser exclusion du bleu trypan Note: Entre quatre jours et six jours de différenciation, EBS cavitation qui se traduit par une grande quantité de l&#39;apoptose et death.Thus cellule, à six jours, plus de 30% des EB cellules dérivées peuvent tache avec du bleu trypan. </li><li> Resuspendre les cellules dérivées de EB à 100.000 cellules / 2 ml de 10% IMDM + virale surnageant tel que une MOI de 5-10 est achieved.Add sulfate de protamine à une concentration finale de 8 mg / mL. Note: Préparer assez de EB / viraux mélange de surnageant à l&#39;assiette quatre plaques à 6 puits (en supposant que 2 ml / puits). </li><li> Planche 2 ml EB / virale mélange surnageant par puits de quatre plaque de 6 puits de pré-plaqué avec des cellules du stroma OP9 (voir ci-dessous). </li><li> Centrifuger les plaques à 2500 rpm à température ambiante pendant 90 minutes. Transfert des plaques d&#39;incubateur à 37 ° C 5% de CO 2. </li><li> 4-6 heures plus tard, la récolte surnageant de plaques et de collecter toutes les cellules potentielles restant en suspension par centrifugation. </li><li> Resuspendre le culot (peut être de petite taille) dans 48 ml IMDM 10% + cytokines. Distribuer 2 ml / puits de remise en suspension à l&#39;initial de quatre plaques de 6 puits dans lequel l&#39;infection a été effectuée et le surnageant a été recueilli. Remarque: Toujours préparer 10% IMDM + cytokines frais au moment de leur utilisation. </li><li> Incuber les plaques à 37 ° C CO 2 5% pour les sept jours. Colonies devrait être évident après l&#39;infection de quatre jours et grandes et bien formé par sept jours. Une forte expansion devrait donner 40 à 60 colonies / puits sur sept jours. </li><li> Sur sept jours post-infection, de collecter et regrouper toutes les cellules (y compris OP9 stroma) de tous les puits par un traitement à la trypsine. Remarque: Ne pas jeter le surnageant ou PBS lave employées durant la trypsine treatment.At ce point dans la culture, certaines colonies peuvent être que faiblement adhérentes et il ya de nombreuses cellules floaTing dans suspension.Collect et la piscine tous les lavages et un surnageant afin de s&#39;assurer qu&#39;aucun des cellules potentiellement précieuses sont jetés. </li><li> Resuspendre les cellules dans 8 ml IMDM 10% + frais de cytokines. Distribuer 2 ml / flacon en quatre flacons T75. Ajouter une supplémentaire de 13 ml + 10% cytokines IMDM à chaque flacon. Incuber à 37 ° C 5% CO 2 pendant trois jours (soit un total de dix jours après l&#39;infection). </li></ol> Culture et le placage de OP9 stroma <ol><li> Maintenir OP9 stroma selon des protocoles standard de 20% a-MEM. OP9 cellules va changer les propriétés lorsqu&#39;il est cultivé jusqu&#39;à la confluence. Toujours diviser à 80% de confluence à pas plus de 1h03 divisée. </li><li> 24 heures avant l&#39;infection des six jours EB cellules dérivées d&#39;MSCV-HOXB4-IRES-GFP, plaque 25000 OP9 cellules / puits de quatre plaques de 6 puits dans 20% une MEM-. </li></ol> Collection, le fractionnement et la transplantation <ol><li> Recueillir les cellules du stroma OP9 élargi sur une dizaine de jours par traitement à la trypsine. Compter les cellules. Remarque: Ne pas jeter le surnageant ou PBS lave employée pendant le traitement trypsine. À ce stade de la culture, certaines colonies peuvent être que faiblement adhérentes et il ya beaucoup de cellules flottant dans suspension.Collect et la piscine tous les lavages et un surnageant afin de s&#39;assurer qu&#39;aucun des cellules potentiellement précieuses sont jetés. Note: Une bonne expansion devrait donner entre 40-50 x 10 6 cellules / 6 original bien-plaque infectés, pour un total de 160-200 x 10 6 cellules. </li><li> Les cellules peuvent être fractionnés par sélection billes magnétiques ou FACS selon des techniques standard à ce point dans le protocole. </li><li> Pour la transplantation, de livrer les cellules via rétro-orbitaires ou injection veine de la queue d&#39;Rag-2/gc destinataires déficiente (pesant entre 15-22 grammes) soumis à une dose de 9,25 Gy diviser d&#39;irradiation 2,5 heures. de l&#39;autre. Note: Il est essentiel que le poids des animaux tombent entre 15-22 grammes au moment de l&#39;irradiation. Si elles sont plus grandes que 22 grammes, 9,25 Gy ne sera pas une dose suffisante de l&#39;irradiation pour assurer l&#39;ablation appropriées et les cellules ne peuvent pas servir de médiateur de sauvetage de l&#39;irradiation létale et / ou se greffer le compartiment hématopoïétique. Si la transplantation de plus grands animaux, la dose d&#39;irradiation approprié doit être déterminée expérimentalement. </li><li> Une dose minimale de 2-5 x 10 6 cellules / animal est nécessaire pour garantir le sauvetage de l&#39;irradiation mortelle. Si le fractionnement des cellules, injecter 2-5 x 10 6 cellules équivalents (par exemple si la population d&#39;intérêt représente 10% du pool de cellules non fractionnée, injecter 2-5 x 10 5 cellules / animal). Remarque: Si l&#39;injection de&gt; 2 x 10 6 cellules, TOUJOURS injectent via veine de la queue pour éviter la formation de tératome en orbite, ce qui peut se produire lorsque le nombre élevé de cellules sont livrés rétro-orbital. </li><li> Maintenir Rag-2 - / - / gc - / - déficiente destinataires dans des cages en autoclave. Selon le «propreté» de l&#39;animalerie, post-transplantation d&#39;animaux peut nécessiter l&#39;administration de l&#39;eau acidifiée ou Trimethiprim-Sulfasoxazole (Septra) pour les 5 semaines suivant le rayonnement mortel. </li><li> Attendez-vous&gt; GFP + 90% ES dérivées de leucocytes dans le sang périphérique à 3 semaines post-transplantation. La prise de greffe doit être multi-lignée, même si les lymphocytes sont connus pour réduire au silence rétroviraux HOXB4-IRES-GFP. </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C57Bl/6 Rag-2/gamma-c deficient mice</td>
<td>Animal</td>
<td>Taconic Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>VWR</td>
<td>33998-360</td>
<td>Orbital Shaker by Ikaworks</td>
</tr>
<tr>
<td>Multi-channel pipettor</td>
<td>Tool</td>
<td>VWR</td>
<td>15000-174</td>
<td>ePet by BioHit</td>
</tr>
<tr>
<td>ES trypsin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>0.25% Trypsin in PBS</td>
</tr>
<tr>
<td>Standard trypsin</td>
<td>Reagent</td>
<td>Invtrogen</td>
<td>25300-062</td>
<td>0.05% Trypsin-EDTA</td>
</tr>
<tr>
<td>PBS</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Enzyme-free dissociation buffer</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>13151-014</td>
<td>&nbsp;</td>
<tr>
<td>Dissociation enzyme mix</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>500 mg Collagenase IV</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H2126</td>
<td>freeze in 500 uL aliquots and avoid repeated freezing and thawing.</td>
</tr>
<tr>
<td>DNAse</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D4527</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10-017CV</td>
<td>&nbsp;</td>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3369</td>
<td>8 ug/mL</td>
</tr>
<tr>
<td>EB differentiation media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM+cytokines</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>20% anti-MEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>		</tbody>
		</table>
		</div>

derivation of hematopoietic stem cells from murine embryonic stem cells

Une cellule souche est définie comme une cellule avec la capacité à la fois à l&#39;auto-renouvellement et de générer de multiples descendance différenciée. Les cellules souches embryonnaires (CSE) sont dérivées de la blastocyste de l&#39;embryon précoce et sont pluripotentes dans la capacité de différenciation. Leur potentiel de différenciation majorité en a fait l&#39;objet de nombreuses recherches centrées sur la façon de les déduire coaxial pour générer des types cellulaires cliniquement utiles. La dérivation réussie de cellules souches hématopoïétiques (CSH) de la souris ESC a récemment été accompli et peuvent être visualisées dans ce protocole vidéo. HSC, sans doute la population cellulaire la plus cliniquement exploitées, sont utilisés pour traiter une multitude de cancers hématopoïétiques et troubles. Toutefois, de nombreux patients qui pourraient bénéficier d&#39;un traitement HSC n&#39;ont pas accès aux bailleurs de fonds appropriés. ESC pourrait fournir une source alternative de HSC pour ces patients. Le protocole suivant établit une base à partir desquelles l&#39;ESC-HSC peuvent être étudiés et les efforts visant à isoler les CSH de CSE humaines. Dans ce protocole, l&#39;ESC sont différenciées comme des corps embryoïdes (EBS) pour 6 jours dans le sérum disponible dans le commerce présélectionnés pour la différenciation hématopoïétique optimale. EB sont alors dissociées et infectés par HOXB4 rétroviraux. Infected EB cellules dérivées sont étalées sur OP9 stroma de la moelle osseuse de cellules stromales dérivées de la ligne de la calvaria de M-CSF / - souris, et co-cultivées en présence de l&#39;hématopoïèse promotion des cytokines pendant dix jours. Lors de cette co-culture, les cellules infectées connaissent une forte expansion, ce qui entraîne la génération d&#39;une piscine hétérogène de 100s de millions de cellules. Ces cellules peuvent ensuite être utilisées à des souris de sauvetage et de reconstituer une irradiation létale.

Watch this Scientific Journal Video about Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells at JoVE.com

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Ce protocole décrit la dérivation de cellules souches hématopoïétiques transplantables à partir des cellules souches embryonnaires de souris (CES) et leur injection ultérieure dans des souris irradiées de façon létale bénéficiaires. Brièvement, l&#39;ESC sont différenciées comme des corps embryoïdes, qui sont ensuite infectées par HOXB4 rétroviraux et co-cultivées avec des cellules stromales et OP9 cytokines hématopoïétiques.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

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 ...

derivation-hematopoietic-stem-cells-from-murine-embryonic-stem

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JoVE Journal

Biology

13.2K vues.  Harvard Medical School. Ce protocole décrit la dérivation de cellules souches hématopoïétiques transplantables à partir des cellules souches embryonnaires de souris (CES) et leur injection ultérieure dans des souris irradiées de façon létale bénéficiaires. Brièvement, l'ESC sont différenciées comme des corps embryoïdes, qui sont ensuite infectées par HOXB4 rétroviraux et co-cultivées avec des cellules stromales et OP9 cytokines hématopoïétiques.

Vidéo: Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagation des souches embryonnaires humaines (ES) Cellules

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Biologie

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

L&#39;isolement et la transplantation de cellules souches hématopoïétiques (CSH)

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.

Dérivation des cellules souches embryonnaires humaines par 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 Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolement et analyse de cellules souches hématopoïétiques à partir du placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

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

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

L&#39;isolement des premiers cellules souches hématopoïétiques de murin sac vitellin et AGA

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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;isolement et la dérivation de cellules germinales embryonnaires de souris

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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.

Dérivation de cellules souches de la souris Trophoblast à partir de blastocystes

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

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.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

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 ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Analyse phénotypique et isolement de cellules souches hématopoïétiques murines et Lineage-engagés progéniteurs

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...