Embryonnaires humaines études de cellules souches dans le laboratoire Teitell sont actuellement pris en charge par une California Institute for Regenerative Medicine (CIRM) Subvention de démarrage RS1-00313. Nous remercions le Dr Carla Koehler (UCLA) et des membres du laboratoire Koehler de fournir des conseils et des idées dans ces domaines et des études supplémentaires.

Haute Résolution électrophorèse sur gel natif Effacer (hrCNE) pour le complexe mitochondrial tests d&#39;activité dans le gel Cellules souches embryonnaires humaines (CSEh) ont été maintenus sur une monocouche de ˠ-irradiés ou la mitomycine C fibroblastes de souris traitées embryonnaires (MEF), puis passer à la croissance sur Matrigel pendant 5 jours dans MEF milieu conditionné utilisant des conditions standard. Pour enlever la contamination FAE, CSEh ont été re-plaquées sur Matrigel et cultivées en milieu conditionné MEF pour les 3 jours supplémentaires. Immunofluorescence pour les marqueurs de pluripotence Oct-4, SSEA-4 a été utilisé pour confirmer l&#39;absence de différenciation des CSEh. <ol><li> Laver les cellules avec des chaudes 1x PBS, pH 7,4, suivis par trypsinisation (1ml de la trypsine 1x par puits dans une plaque à 6 puits) pendant 5 min. </li><li> Ajouter un volume égal d&#39;inhibiteur de trypsine frais (1mg/ml dans du PBS 1X, pH 7,4). Récolte des cellules et centrifuger à 200 g, 5 min, la température ambiante. </li><li> Jeter le surnageant et remettre les cellules granulées de 0,8 - 1.7ml (basée sur la taille du culot cellulaire) de glace isolée tampon froid mitochondries (83mm saccharose; 6.6mm imidazole / HCl, pH 7,0) avec 1 fraîchement préparés &quot;cocktail inhibiteur de protéase. Incuber sur la glace pendant 10 min. </li><li> Dounce cellules avec un homogénéisateur (~ 40 - 50 coups). Stain une aliquote de cellules dounced bleu trypan pour vérifier l&#39;exhaustivité de l&#39;homogénéisation à l&#39;aide d&#39;un microscope optique. </li><li> Centrifuger à 20000 g pendant 10 min à 4 ° C pour obtenir un brut mitochondrial granulés qui contient également des noyaux et des fragments de cellules plus grandes. Décanter et jeter le surnageant et peser le précipité brut mitochondrial granulés. Le culot peut être conservé à -80 ° C après la congélation instantanée dans l&#39;azote liquide, si ce n&#39;est un point d&#39;arrêt. REMARQUE: centrifugation différentielle ou d&#39;autres basées sur les gradients procédures peuvent être utilisées pour isoler et de percevoir une fraction de membrane mitochondriale pures. Voir réf. 4 pour les détails. Cette approche alternative sera le rendement de l&#39;activité mitochondriale complexes par mg de protéine mitochondriale lieu d&#39;activité complexe par mg de poids cellulaire ou un numéro de cellulaire. La concentration de protéines mitochondriales devra être déterminé après la purification de faire cette comparaison. En outre, un tampon d&#39;isolement différent peut être requis pour cette approche. Pour la solubilisation de la fraction de la membrane mitochondriale pur, voir la note suivante étape 6 ci-dessous. </li><li> Solubiliser le brut mitochondrial granulés sur la glace au vortex avec un tampon de solubilisation de la glace froide (50 mM NaCl, 50 mM imidazole / HCl pH 7,0, 1 mM EDTA, 2 mM d&#39;acide 6-aminohexanoïque). L&#39;acide 6-aminohexanoïque peut être remplacé par le cocktail d&#39;inhibiteur de protéase 1x. Utilisez 35μl de tampon de solubilisation par 20mg de poids à granulés. Ajouter 20 pi de 10% p / v digitonine par 20mg de poids granulés et mélanger en feuilletant le tube. Incuber 5-10 min sur la glace. Dodécyl-bD-maltoside ou le Triton X-100 peut être substitué à la digitonine pour solubiliser les protéines. Le choix du détergent et de sa quantité peuvent affecter la formation de formes multimériques supercomplexes ou des complexes mitochondriaux. Pour plus d&#39;informations, consultez ref.1. REMARQUE: La quantité de détergent nécessaire pour solubiliser une fraction membranaire mitochondrial pur correspond à un ratio poids du détergent / protéine allant de 2 à 6g / g. Le rapport optimal pour solubiliser les membranes mitochondriales est 2.5g / g pour dodécyl-bD-maltoside, 3g / g pour le triton X-100, et 6g / g pour la digitonine. </li><li> Centrifuger le culot solubilisé à 100.000 g pendant 15 min à 4 ° C. Recueillir le surnageant clair. </li><li> Ajouter un tampon de charge à une finale de 5% p / v de glycérol et 0,01% p / v Ponceau S (utilisation de 50% p / v de glycérol, 0,1% w / v de solution de chargement Ponceau S stock tampon). </li><li> Charger des puits du gel avec un volume qui contient 20-40mg du brut culot de mitochondries par voie. Utilisez un gel à gradient d&#39;acrylamide (4 -13%) avec un empilage de 3,5% de gel. REMARQUE: préparation de gel et les conditions de l&#39;électrophorèse: Pour la préparation du gel, utiliser un acrylamide / bis-acrylamide (AA / bisAA) 3 mélanger comme suit: 48g d&#39;acrylamide et 1,5 g de bisacrylamide (49,5% T, 3% C. Si T est la concentration totale d&#39;AA et bisAA et C est le pourcentage de réticulant (bisAA) au total des monomères). Gel de tampon (3 &#39;): 1,5 M acide 6-aminohexanoïque; 75mm imidazole, pH 7,0 </li></ol><table style="padding-left: 60px;" border="0"><tbody><tr><td> Gel de gradient: </td><td> 4% </td><td> 13% </td><td> Empilement de gel: </td><td> 3,5% </td></tr><tr><td> AA / 3 mix bisAA </td><td> 2 ml </td><td> 6,5 ml </td><td></td><td> 0,6 ml </td></tr><tr><td> Gel de tampon 3 &#39; </td><td> 8,3 ml </td><td> 8,3 ml </td><td></td><td> 2,7 ml </td></tr><tr><td> Glycérol </td><td></td><td> 5 g </td><td></td><td></td></tr><tr><td> H 2 O </td><td> 14,7 ml </td><td> 5 ml </td><td></td><td> 4,7 ml </td></tr><tr><td> Le volume total </td><td> 25 ml </td><td> 25 ml </td><td></td><td> 8 ml </td></tr><tr><td> APS 10% </td><td> 139 ul </td><td> 125 pi </td><td></td><td> 67 ul </td></tr><tr><td> TEMED </td><td> 14 ul </td><td> 12,5 ul </td><td></td><td> 6,7 ul </td></tr></tbody></table> Anode tampon: 25mm imidazole / HCl, pH 7,0 Buffer Cathode: Tricine 50 mM, 7,5 mm d&#39;imidazole / HCl, 0,02% p / v dodécyl-bD-maltoside, 0,05% p / v désoxycholate, pH 7,0 Verser et exécuter des gels dans une chambre froide (4-7 ° C). La tension initiale utilisée est de 100V. Lorsque l&#39;échantillon est entré dans le gel de concentration, la tension est élevée à 500V avec le courant limité à 15mA (pour 0,15 &#39;14&#39; 14 cm gels). L&#39;électrophorèse est arrêté après 3-4 h quand la ligne forte de colorant du rouge Ponceau S se rapproche de la première gel. Alternativement, un gel peut être exécuté pendant la nuit avec une tension constante à 70-100V. <ol><li> Tests d&#39;activité dans le gel. Le temps d&#39;incubation pour chaque dosage dépend de l&#39;efficacité de la solubilisation des protéines et la quantité de matière chargée par puits. Complexes I, IV et V donnent habituellement des signaux plus forts que des complexes II et III. </li></ol> Complexe I (CI) Incuber la tranche de gel avec CI tampon (Tris-HCl 5 mM, pH 7,4; 0.1mg/ml NADH; 2.5mg/ml NBT) à température ambiante pendant plusieurs heures. CI mémoire tampon: Ajouter de 5 mm par 100 ml de Tris-HCl, pH 7,4: 0,01 g NADH 0.25g NBT Fixer le gel dans du méthanol à 50% et 10% d&#39;acide acétique pour les 15-45 min. Le gel fixe est conservé dans l&#39;acide acétique à 10%. Complexe II (CII) Incuber la tranche de gel avec des CII tampon (20 mM de sodium succinate; methasulfate phénazine 0.2mm; 2.5mg/ml NBT; 5mM Tris-HCl pH 7,4) à température ambiante pendant plusieurs heures. CII mémoire tampon: Ajouter de 5 mm par 100 ml de Tris-HCl, pH 7,4: 200 pl de succinate de sodium 1M Methasulfate phénazine 80μl (PMS 250mm dissous dans du DMSO) 0.25g NBT Fixer le gel comme décrit pour le complexe I Complexe III (CIII) Incuber la tranche de gel avec CIII tampon (phosphate de sodium 50 mM, pH 7,2, de 0,05% de 3,3 &#39;-diaminobenzidine tétrachlorhydrate (DAB)) à température ambiante pendant plusieurs heures. CIII tampon: Ajouter par 100 ml de phosphate de sodium 50 mM, pH 7,2: Tétrachlorhydrate diaminobenzidine 50mg Fixer le gel comme décrit pour le complexe I Complexe IV (CIV) Incuber la tranche de gel avec CIV tampon (phosphate de sodium 50 mM, pH 7,2, de 0,05% de 3,3 &#39;-diaminobenzidine tétrachlorhydrate (DAB), 50 microns cheval de coeur du cytochrome c) à température ambiante pendant plusieurs heures. CIV tampon: Ajouter par 100 ml de phosphate de sodium 50 mM, pH 7,2: Tétrachlorhydrate diaminobenzidine 50mg 0.1g cheval de coeur du cytochrome c Fixer le gel comme décrit pour le complexe I Complexe V (CV) Incuber la tranche de gel avec CV de tampon (Tris-35mm de base, 270mm de glycine, pH 8,3 à 25 ° C, 14mm MgSO 4, 0,2% p / v Pb (NO 3) 2, 8mm ATP) à température ambiante pendant plusieurs heures. CV de tampon: 100ml de solution par ajouter: <table border="0"><tbody><tr><td> Base Tris </td><td> 0.424g </td></tr><tr><td> Glycine </td><td> 2.026g </td></tr><tr><td> MgSO 4 </td><td> 0.345g </td></tr><tr><td> Pb (NO 3) 2 </td><td> 0.29g </td></tr></tbody></table> Pas besoin d&#39;ajuster le pH (qui devrait être 8.3) - ne pas utiliser d&#39;acide ou de base. Filtrez et ajoutez <table border="0"><tbody><tr><td> ATP </td><td> 0.441g </td></tr></tbody></table> Fixer le gel dans du méthanol à 50% pendant 30 minutes et le transfert à l&#39;eau.

<ol>
	<li>Wittig, I., Braun, H. -. P., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nature Protocols. 1, 418-428 (2006).</li><li>Wittig, I., Karas, M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Resolution+Clear+Native+Electrophoresis+for+In-gel+Functional+Assays+and+Fluorescence+Studies+of+Membrane+Protein+Complexes.++6.">High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes. 6.</a> Molecular &amp; Cellular Proteomics. 6, 1215-1225 (2007).</li><li>Wittig, I., Carrozzo, R., Santorelli, F. M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assays+in+high-resolution+clear+native+gels+to+quantify+mitochondrial+complexes+in+human+biopsies+and+cell+lines.">Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.</a> Electrophoresis. 28, 3811-3820 (2007).</li><li>Castle, J. D., Coligan, J. E., Dunn, B. M., Speicher, D. W., Wingfield, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Organelles+from+Mammalian+Cells.">Purification of Organelles from Mammalian Cells.</a> Current Protocols in Protein Science. 4.2, 4.2.1-4.2.57 (2007).</li></ol>

Dans cette vidéo, nous avons décrit l&#39;extraction des complexes de protéines mitochondriales de cellules souches embryonnaires humaines, leur séparation par électrophorèse haute résolution claire gel natif (hrCNE), et l&#39;analyse subséquente par en gel des essais d&#39;activité catalytique. hrCNE résout mitochondriale des complexes protéine membranaire à une résolution comparable à celle de l&#39;électrophorèse sur gel bleu natif et est supérieure pour les tests d&#39;activité dans le gel. Cette technique peut être e...

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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>0.5M EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP120-1</td>
<td>adjust pH to 8.0 with NaOH </td>
</tr>
<tr>
<td>1M NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014-500MG</td>
<td>&nbsp;</td>
<tr>
<td>1M Sodium succinate</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>S413-500</td>
<td>&nbsp;</td>
<tr>
<td>0.1M Sodium phosphate buffer pH 7.2</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP332-1 and BP329-1</td>
<td>Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C </td>
</tr>
<tr>
<td>1M Tricine, pH 7.0 (adjusted with NaOH)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>9010</td>
<td>store at 4 C </td>
</tr>
<tr>
<td>1M imidazole/HCl, pH 7.0</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-2399</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>1M Tris-HCl, pH 7.4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP153-1</td>
<td>&nbsp;</td>
<tr>
<td>20% (wt/vol) dodecyl-b-D-maltoside (DDM)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>44205-500MG</td>
<td>Dissolved in water. Store 1 ml aliquots at &#x2013;20 C.</td>
</tr>
<tr>
<td>10% (wt/vol) digitonin (>50% purity, used without recrystallization)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>37008-1G</td>
<td>Dissolved in water. Store 0.1&#x2013;1 ml aliquots at &#x2013;20 C. Should be warmed up to 95 C before use.</td>
</tr>
<tr>
<td>2M 6-aminohexanoic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>07260-100G</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>10x Ponceau S/glycerol stock solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3504 and G6279-1L</td>
<td>0.1% Ponceau S, 50% glycerol, (wt/vol) in water.</td>
</tr>
<tr>
<td>10% w/v sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Fluka </td>
<td>30970-25G</td>
<td>Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature.</td>
</tr>
<tr>
<td>250 mM phenazine methasulfate (PMS)</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>P0083</td>
<td>Dissolved in DMSO and aliquoted. Store at &#x2013;20 C.</td>
</tr>
<tr>
<td>Nitro-blue tetrazolium (NBT)</td>
<td>&nbsp;</td>
<td>Amresco </td>
<td>0329-1G</td>
<td>&nbsp;</td>
<tr>
<td>3,3&#x2019;-diaminobenzidine tetrahydrochloride (DAB)</td>
<td>&nbsp;</td>
<td>TCI America </td>
<td>D0078</td>
<td>&nbsp;</td>
<tr>
<td>Pb(NO3)2</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>L62-100</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP213-1</td>
<td>&nbsp;</td>
<tr>
<td>Tris base </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP152-10</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>G45-212</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2019;-triphosphate disodium salt (ATP)</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2383-5G</td>
<td>Grade I, minimum 99%</td>
</tr>
<tr>
<td>beta-nicotineamide adenine dinucleotide (NADH) reduced form </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>N-8129</td>
<td>&nbsp;</td>
<tr>
<td>Cytochrome c From horse heart</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C2506-250MG</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP220-212</td>
<td>&nbsp;</td>
<tr>
<td>100x protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P8340</td>
<td>Store at &#x2013; 20 C.</td>
</tr>
<tr>
<td>Dimethyl sulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D128-500</td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue Stain 0.4%</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>10x Trypsin (0.5%)</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15400-054</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>17075-029</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

probing for mitochondrial complex activity in human embryonic stem cells

Les mitochondries sont des organites cytoplasmiques qui ont un rôle primordial dans le métabolisme cellulaire et l&#39;homéostasie, la régulation du réseau de signalisation cellulaire, et la mort cellulaire programmée. Les mitochondries produisent l&#39;ATP, de réglementer l&#39;état redox cytoplasmique et Ca2 + bilan, cataboliser acides gras, de synthétiser l&#39;hème, nucléotides, hormones stéroïdes, acides aminés, et aider à assembler fer-soufre dans les protéines. Les mitochondries jouent également un rôle essentiel dans la production de chaleur. Les mutations du génome mitochondrial entraîner plusieurs types de trouble humain. L&#39;accumulation de mutations de l&#39;ADNmt est en corrélation avec le vieillissement et est soupçonné d&#39;avoir un rôle important dans le développement du cancer. En raison de leur rôle d&#39;une importance vitale dans tous les types de cellules, la fonction des mitochondries doit aussi être critique pour les cellules souches. Avancées majeures ont été faites dans notre compréhension de la viabilité des cellules souches, la prolifération, et la capacité de différenciation. Mais l&#39;activité fonctionnelle des cellules souches, en particulier de leur état énergétique, ne fut pas encore été étudiés en détail. Presque rien n&#39;est connu sur les propriétés des mitochondries des cellules souches embryonnaires humaines (CSEh) et leur descendance différenciée précurseur. Une façon de comprendre et d&#39;évaluer le rôle des mitochondries dans la fonction de CSEh et le potentiel de développement est de mesurer directement l&#39;activité des complexes respiratoires mitochondriaux. Ici, nous décrivons haute résolution électrophorèse sur gel natif claires et les suivants dans la visualisation de l&#39;activité de gel comme une méthode pour analyser les cinq complexes de la chaîne respiratoire des CSEh.

Watch this Scientific Journal Video about Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells at JoVE.com

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Dans cette vidéo, nous allons vous montrer comment les complexes de la chaîne respiratoire mitochondriale des cellules souches embryonnaires humaines peuvent être analysées à l&#39;aide des tests d&#39;activité de gel.

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 ...

probing-for-mitochondrial-complex-activity-human-embryonic-stem

Research

JoVE Journal

Biology

14.2K vues.  University of California, Los Angeles. Dans cette vidéo, nous allons vous montrer comment les complexes de la chaîne respiratoire mitochondriale des cellules souches embryonnaires humaines peuvent être analysées à l'aide des tests d'activité de gel.

Vidéo: Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

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 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 ...

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Marquage in vitro des cellules souches embryonnaires humaines pour l&#39;imagerie par résonance magnétique

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vidéo analyse bioinformatique des droits de croissance de la tige colonie de cellules embryonnaires

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.