AEC est membre de la Jane Coffin Childs Memorial Fund pour la recherche médicale et de laboratoires de recherche Merck. Nous remercions D. Egli et C. Cowan pour des conseils d&#39;expérimentation et de discussion.

<html> Frozen embryons humains utilisés pour la dérivation de cellules souches humaines embryonnaires humaines (hES) ont été donnés pour la recherche après consentement éclairé appropriée en vertu des protocoles de recherche qui ont été examinés et approuvés tant par le Comité sur l&#39;utilisation de sujets humains et les cellules souches embryonnaires de surveillance du comité de recherche à l&#39;Université Harvard . Protocole écrit ci-dessous est basée sur des paramètres publié rapportés par Cowan et al. 2004, avec de légères modifications. Culture des embryons Dégel des embryons humains en utilisant Kit Quinn Avantage dégel et la culture en gouttes de milieu mondiale complétée par Plasmanate 15% jusqu&#39;au développement jusqu&#39;au stade blastocyste élargi. Immunosurgery basée sur l&#39;isolement de la masse cellulaire interne Préparation: <ol><li> Préparer les plaques pour le retrait de la zone pellucide et immunosurgery (voir ci-dessous) en mettant en place des gouttes de chaque solution dans des assiettes de 10 cm. Superposition avec l&#39;huile minérale pour éviter l&#39;évaporation. Préparer une seule rangée par embryon destiné à la dérivation. <ul><li> EmbryoMax Tyrodes acide, utiliser tel quel. </li><li> les médias dérivation de cellules hES: 75% KO-DMEM, 10% KO-Sérum remplacement, Plasmanate 10%, 2,5% des cellules ES testé sérum fœtal bovin (FBS), 2 mM Glutamax-I, 1% de non-acides aminés essentiels, 50 unités / ml de pénicilline, la streptomycine 50μg/ml, 0.055mM b-mercaptoéthanol, 5ng/ml bFGF. </li><li> Lapin anticorps anti-humain de globules rouges primaire, reconstitué selon la suggestion du fabricant, dilué à 1:10 avec les médias dérivation de cellules hES. </li><li> Guinée-sérum de porc complément, reconstitué selon la suggestion du fabricant, dilué à 1:10 avec les médias dérivation de cellules hES. <img src="/files/ftp_upload/old/574_2008_9_18_6/574_Figure-01.png" alt="574_Figure-01" width="600" height="166" /> </li></ul></li><li> Equilibrer toutes les plaques à 37 ° C en incubateur de culture tissulaire avant utilisation. </li><li> Tirez 50 pl-100 ul pipettes capillaires (VWR) pour une utilisation dans des embryons de transfert. Extrémités coupées à la plume de diamants et de l&#39;utilisation avec des tubes pipeter à la bouche équipé d&#39;un filtre 0,2 um. </li></ol> Procédure Immunosurgery: Notes: <ul><li> Dans cette procédure, les cellules du trophectoderme sont détruits par une brève exposition à des anticorps dirigés contre les cellules humaines en tandem avec l&#39;activité du complément. Par conséquent, le protocole fonctionne mieux avec embryons de grande qualité avec un trophectoderme intacte, comme seule l&#39;intégrité structurale du blastocyste empêche l&#39;ICM d&#39;être aussi sensibles à la réaction immunologique. </li><li> Toutes les procédures effectuées en utilisant un champ de dissection équipés de platine chauffante. </li></ul> Étapes: <ol><li> Rincer la pipette bouche pour être utilisé pour le transfert d&#39;embryons avec du FBS embryons pour éviter de coller. </li><li> Transfert blastocyste de boîte de culture pour la tenue hES goutte d&#39;AT plat. </li><li> Début de la procédure par le transfert de blastocyste de déposer hES grâce à une série d&#39;AT gouttes. En troisième baisse, montre pour la dissolution de la zone pellucide (secondes en général 5-30). Attention à ne pas l&#39;intégrité sur-traiter ou de l&#39;embryon peut être compromise. <img src="/files/ftp_upload/old/574_2008_7_21_2/574_Figure-02.png" alt="574_Figure-02" width="551" height="273" /> </li><li> Blastocyste transfert grâce à une série de médias dérivation de cellules hES gouttes de rincer à l&#39;arrêt. Pipette préchargement avec les médias est recommandé de neutraliser immédiatement la réaction. Blastocystes peut être tenu ici jusqu&#39;à ce que tous ont été au-traitées. </li><li> Transfert blastocyste grâce à une série de gouttes d&#39;anticorps primaires, laissant en troisième baisse pour 30 minutes d&#39;incubation à 37 ° C dans un incubateur de culture tissulaire. </li><li> Blastocyste transfert grâce à une série de médias dérivation de cellules hES tombe à rincer primaire. </li><li> Transfert blastocyste grâce à une série de gouttes complètent, en laissant au troisième baisse de regarder pour la lyse des cellules du trophectoderme. Observez blastocyste pour les 30 premières secondes sur scène pour &quot;bouillonnante&quot; des cellules du trophectoderme comme ils lysent. Si la lyse est observée dans ce délai, gardez plat sur la platine du microscope pour surveiller la réaction jusqu&#39;à son achèvement. Si aucun bullage est observé pendant les 30 premières secondes, retour à la pépinière, en vérifiant toutes les 5 minutes jusqu&#39;à ce que la plupart des trophectoderme a lysées. Cela peut prendre 5-15 minutes. Surveiller de près la réaction afin de minimiser le temps de réaction et les dommages potentiels à la masse cellulaire interne. <img src="/files/ftp_upload/old/574_2008_7_21_3/574_Figure-03.png" alt="574_Figure-03" width="571" height="273" /> </li><li> Blastocyste transfert grâce à une série de médias dérivation de cellules hES gouttes pour laver l&#39;activité du complément. </li><li> En utilisant une pipette en verre avec un diamètre qui est légèrement plus petit que le blastocyste (tirée de tubes capillaires VWR 10 ul), dessiner le blastocyste haut et bas pour dépouiller la plupart des trophectoderme lysées. g &quot;alt =&quot; 574_Figure-0 &quot;width =&quot; 577 &quot;height =&quot; 270 &quot;/&gt; </li><li> Plaque d&#39;isoler l&#39;ICM sur les cellules nourricières de la mitose des fibroblastes d&#39;embryon de souris inactivées (FAE). FAE devrait être préparé la veille et sont passés à la dérivation de cellules hES dérivation journée des médias d&#39;. 4-plats ainsi NUNC fonctionnent bien pour des mesures initiales de la dérivation. </li><li> Ne pas déranger la plaque pour la première 1-2 jours. Par la suite, chaque jour vérifier excroissance de cellules souches embryonnaires colonies (généralement apparente dans 1-2 semaines). Les médias devraient être changés dans les volumes moitié tous les deux jours commence le jour 3. </li></ol> Excroissance de cellules ES et repiquage Lorsque l&#39;excroissance de cellules hES a atteint environ 0,5 mm ou plus, il est prêt pour l&#39;expansion par la cueillette mécanique. <ol><li> Mécaniquement disperser la moitié de l&#39;excroissance et transfert à de nouvelles plaques contenant mangeoires frais. Touffes de cellules souches embryonnaires doivent être d&#39;environ 30-50 cellules chacune. Laisser l&#39;autre moitié d&#39;excroissance comme un back-up pour le premier passage (P1). L&#39;excroissance (p0) peut être capté à partir plusieurs fois car il continue à se développer. <img src="/files/ftp_upload/old/574_2008_7_21_5/574_Figure-05.png" alt="574_Figure" width="639" height="219" /> </li><li> Colonies secondaires peuvent être également dispersées et étendu à l&#39;ensemble des boîtes de culture plus que les passages 3-5, époque à laquelle les lignées cellulaires de nouvelles peuvent être adaptées aux repiquage enzymatique à la trypsine. Le contenu du sérum des milieux de culture doivent également être progressivement ramenée à 1% et 0% au cours de ces passages initiaux afin de minimiser la différenciation des cellules ES. </li><li> Tous établie lignées cellulaires embryonnaires souches devraient être mis en réserve par le gel jusqu&#39;à une partie de passages tôt pour de futures expansions. </li><li> En outre, chaque lignée cellulaire devrait être caractérisé par l&#39;analyse du caryotype et validé par des études de pluripotence et différenciation in vitro et in vivo. </li></ol>

<ol>
	<li>Cowan, C. A., Klimanskaya, I., McMahon, J., Atienza, J., Whitmyer, J., Zucker, J. P., Wang, S. P., Morton, C. C., McMahon, A. P., Powers, D., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+embryonic+stem-cell+lines+from+human+blastocysts.">Derivation of embryonic stem-cell lines from human blastocysts.</a> N ENGL J MED. 350 (13), 1353-1356 (2004).</li></ol>

The authors have nothing to disclose.

Le protocole présenté ici fournit aux chercheurs une présentation concise, facile à suivre aperçu de la façon de dériver des lignées cellulaires embryonnaires souches par l&#39;isolement immunosurgical de la masse cellulaire interne.

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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>Quinn's Advantage Thaw Kit </td>
<td>&nbsp;</td>
<td>Sage</td>
<td>ART-8016</td>
<td>&nbsp;</td>
<tr>
<td>Global medium</td>
<td>&nbsp;</td>
<td>Life Global</td>
<td>GMGB-050</td>
<td>&nbsp;</td>
<tr>
<td>Plasmanate</td>
<td>&nbsp;</td>
<td>Talecris</td>
<td>61325</td>
<td>&nbsp;</td>
<tr>
<td>EmbryoMax Acidic Tyrodes</td>
<td>&nbsp;</td>
<td>Chemicon/Millipore</td>
<td>MR-004-D</td>
<td>&nbsp;</td>
<tr>
<td>KO-DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>10829-018</td>
<td>&nbsp;</td>
<tr>
<td>KO-Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>ES cell-tested fetal bovine serum, Defined</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30070.03</td>
<td>&nbsp;</td>
<tr>
<td>Glutamax-I</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>35050-079</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>11140-076</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15070-063</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human basic fibroblast growth factor</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256-029</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol </td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-human red blood cell primary antibody</td>
<td>&nbsp;</td>
<td>Rockland</td>
<td>109-4139</td>
<td>&nbsp;</td>
<tr>
<td>Guinea-pig complement serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-1639</td>
<td>&nbsp;</td>
<tr>
<td>Capillary pipettes in 10 microL, 50 microL, 100 microL sizes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouth pipettes with tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>supplied with capillary pipettes from VWR</td>
</tr>
<tr>
<td>0.2 microm Acrodisc syringe filter </td>
<td>&nbsp;</td>
<td>Pall Life Sciences</td>
<td>PN4192</td>
<td>&nbsp;</td>
<tr>
<td>0.5% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>25300-120</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>NUNC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nikon SMZ1500 dissection scope equipped with Nikon Tokai Hit ThermoPlate warming stage </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouse embryonic fibroblasts (E12.5)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

derivation of human embryonic stem cells by immunosurgery

La capacité des cellules souches embryonnaires humaines à se renouveler et se différencier en tous les types cellulaires du corps donne à penser qu&#39;ils sont très prometteurs pour les applications médicales et comme un outil de recherche pour aborder les questions fondamentales dans le développement et la maladie. Ici, nous fournissons un résumé concis, étape par étape de protocole pour la dérivation de cellules souches embryonnaires humaines à partir d&#39;embryons par l&#39;isolement immunosurgical de la masse cellulaire interne.

Watch this Scientific Journal Video about Derivation of Human Embryonic Stem Cells by Immunosurgery at JoVE.com

Derivation of Human Embryonic Stem Cells by Immunosurgery

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 ...

derivation-of-human-embryonic-stem-cells-by-immunosurgery

Research

JoVE Journal

Biology

33.7K vues.  Harvard. La capacité des cellules souches embryonnaires humaines à se renouveler et se différencier en tous les types cellulaires du corps donne à penser qu'ils sont très prometteurs pour les applications médicales et comme un outil de recherche pour aborder les questions fondamentales dans le développement et la maladie. Ici, nous fournissons un résumé concis, étape par étape de protocole pour la dérivation de cellules souches embryonnaires humaines à partir d&#3...

Vidéo: Derivation of Human Embryonic Stem Cells by Immunosurgery

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagation des souches embryonnaires humaines (ES) Cellules

Recherche

Biologie

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.

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Palpage pour activité complexe mitochondrial dans les cellules souches embryonnaires humaines

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Culture et entretien des cellules souches embryonnaires humaines

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Préparation Étaler chromosomique des cellules souches embryonnaires humaines pour le caryotype

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congélation et de décongélation cellules souches embryonnaires humaines

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La différenciation des cellules souches embryonnaires en précurseurs d&#39;oligodendrocytes

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...