Embrionali umane studi sulle cellule staminali in laboratorio Teitell sono attualmente supportati da un Istituto della California per medicina rigeneratrice (CIRM) sementi concedere RS1-00313. Ringraziamo il Dott. Carla Koehler (UCLA) e membri del laboratorio Koehler per fornire consigli e approfondimenti in questi e altri studi.

Alta Electophoresis risoluzione nativa gel trasparente (hrCNE) per complessi mitocondriale In-Gel Attività saggi Cellule staminali embrionali umane (hESC) sono stati mantenuti su un monostrato di ˠ-irradiati o mitomicina C trattati con fibroblasti embrionali di topo (MEF), poi passati a una crescita su Matrigel per 5 giorni in MEF condizionata media con condizioni standard. Per rimuovere i contaminanti MEF, hESC sono stati ri-placcato su Matrigel e cresciuto in MEF condizionata medio per altri 3 giorni. Microscopia di immunofluorescenza per i marcatori pluripotenza Oct-4 e SSE-4 è stato utilizzato per confermare la mancanza di differenziazione hESC. <ol><li> Lavare le cellule con caldo 1x PBS, pH 7,4, seguita da tripsinizzazione (1x 1 ml di tripsina per bene in un 6-pozzetti) per 5 min. </li><li> Aggiungere un uguale volume di fresca inibitore della tripsina (1mg/ml in 1x PBS, pH 7,4). Celle di raccolta e centrifugare a 200g, 5 min, temperatura ambiente. </li><li> Eliminare le cellule surnatante e risospendere pellet in 0,8 - 1.7ml (in base alle dimensioni del pellet) di tampone di ghiaccio freddo isolamento dei mitocondri (83mm saccarosio; 6,6 mm imidazolo / HCl, pH 7,0) con appena preparato 1 &#39;Cocktail di inibitori della proteasi. Incubare in ghiaccio per 10 minuti. </li><li> Dounce cellule con un omogeneizzatore (~ 40 - 50 colpi). Macchia una aliquota di cellule dounced con trypan blu per verificare la completezza di omogeneizzazione utilizzando un microscopio ottico. </li><li> Centrifugare a 20.000 g per 10 minuti a 4 ° C per ottenere un grezzo mitocondriale pellet che contiene anche i nuclei e frammenti di cellule più grandi. Decantare e scartare il surnatante e pesare il greggio precipitato a pellet mitocondriale. Il pellet può essere conservato a -80 ° C a seguito immediato congelamento in azoto liquido, se questo è un punto di arresto. NOTA: centrifugazione differenziale o altri gradiente basato su procedure possono essere utilizzate per l&#39;isolamento e la raccolta di una frazione pura membrana mitocondriale. Vedi rif. 4 per i dettagli. Questo approccio alternativo produrrà l&#39;attività mitocondriale complesso per mg di proteina mitocondriale invece di complessa attività per mg di peso cellulare o numero di cellulare. La concentrazione di proteine ​​mitocondriali dovrà essere determinato dopo la purificazione per fare questo confronto. Inoltre, un buffer di isolamento diversi possono essere richiesti per questo approccio. Per la solubilizzazione della frazione pura membrana mitocondriale, vedere la nota seguente punto 6. </li><li> Solubilizzare il greggio mitocondriale pellet su ghiaccio con il vortex con il tampone di solubilizzazione ghiaccio freddo (50 mM NaCl, 50 mM imidazolo / HCl pH 7.0, 1mM EDTA, 2mM 6-aminohexanoic acido). L&#39;acido 6-aminohexanoic può essere sostituito con il Cocktail 1x inibitore della proteasi. L&#39;uso 35μl di tampone solubilizzazione per 20 mg di peso a pellet. Aggiungere 20μl del 10% w / v digitonina per 20 mg di peso a pellet e mescolare muovendo il tubo. Incubare per 5-10 minuti sul ghiaccio. Dodecil-BD-maltoside o Triton X-100 può essere sostituito per digitonina per solubilizzare le proteine. La scelta del detersivo e la sua quantità può influenzare la formazione di supercomplexes o forme multimerica di complessi mitocondriali. Per ulteriori informazioni, vedere rif.1. NOTA: La quantità di detersivo necessaria per solubilizzare una frazione pura membrana mitocondriale corrisponde ad un detergente / proteine ​​di peso che varia dai 2-6g / g. Il rapporto ottimale per solubilizzare le membrane mitocondriali è 2.5g / g per dodecil-BD-maltoside, 3g / g per Triton X-100, e 6 g / g per digitonina. </li><li> Centrifugare il pellet solubilizzato a 100.000 g per 15 minuti a 4 ° C. Raccogliere il surnatante. </li><li> Aggiungi il caricamento del buffer ad una finale del 5% w / v glicerolo e 0,01% w / v Ponceau S (uso un 50% w / v glicerolo, 0,1% w / v tampone Ponceau S soluzione di carico magazzino). </li><li> Carico pozzi gel con un volume che contiene i 20-40mg del greggio mitocondriale pellet per corsia. Utilizzare un gel gradiente di acrilammide (4 -13%) con un 3,5% stacking gel. NOTA: preparazione gel elettroforesi e condizioni: Per la preparazione di gel, utilizzare un acrilamide / bis-acrilamide (AA / bisAA) 3 miscela come segue: 48 g di acrilamide e 1,5 g di bisacrylamide (49,5% T, 3% C. Se T è la concentrazione totale di AA e bisAA e C è la percentuale di reticolante (bisAA) per monomero totale). Gel tampone (3 &#39;): 1,5 M 6-aminohexanoic acido; 75mm imidazolo, pH 7,0 </li></ol><table style="padding-left: 60px;" border="0"><tbody><tr><td> Gradiente di gel: </td><td> 4% </td><td> 13% </td><td> Stacking gel: </td><td> 3,5% </td></tr><tr><td> AA / bisAA 3 mix </td><td> 2 ml </td><td> 6,5 ml </td><td></td><td> 0,6 ml </td></tr><tr><td> Gel tampone 3 &#39; </td><td> 8,3 ml </td><td> 8,3 ml </td><td></td><td> 2,7 ml </td></tr><tr><td> Glicerina </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> Volume totale </td><td> 25 ml </td><td> 25 ml </td><td></td><td> 8 ml </td></tr><tr><td> 10% di APS </td><td> 139 microlitri </td><td> 125 microlitri </td><td></td><td> 67 microlitri </td></tr><tr><td> TEMED </td><td> 14 microlitri </td><td> 12,5 microlitri </td><td></td><td> 6,7 microlitri </td></tr></tbody></table> Anodo buffer: 25mM imidazolo / HCl, pH 7,0 Catodo buffer: Tricine 50mm, 7.5mm imidazolo / HCl, 0,02% w / v dodecil-BD-maltoside, 0,05% w / v desossicolato, pH 7,0 Versare ed eseguire gel in una stanza fredda (4-7 ° C). La tensione iniziale utilizzato è 100V. Quando il campione è entrato nel gel impilati, la tensione è aumentata a 500V con la corrente limitata a 15mA (per 0,15 &#39;14&#39; 14 cm di gel). Elettroforesi viene arrestata dopo 3-4 ore quando la linea netta di colorante rosso del Ponceau S si avvicina alla parte anteriore gel. In alternativa, un gel può essere eseguito durante la notte con tensione costante a 70-100V. <ol><li> In-gel attività saggi. Il tempo di incubazione per ogni test dipende dall&#39;efficienza di solubilizzazione delle proteine ​​e la quantità di materiale caricato per bene. Complessi I, IV e V di solito resa più forti segnali di complessi II e III. </li></ol> Complesso I (CI) Incubare la fetta gel con CI tampone (5mm Tris-HCl, pH 7.4; 0.1mg/ml NADH; 2.5mg/ml NBT) a temperatura ambiente per diverse ore. IC buffer: Aggiungere per 100 ml di 5mM Tris-HCl, pH 7,4: 0.01g NADH 0,25 g NBT Fissare il gel in metanolo al 50% e 10% di acido acetico per 15-45 min. Il gel fisso è conservata nel 10% di acido acetico. Complesso II (CII) Incubare la fetta gel con CII buffer (20 mM di sodio succinato; methasulfate phenazine 0,2 mm; 2.5mg/ml NBT; 5mM Tris-HCl pH 7,4) a temperatura ambiente per diverse ore. CII buffer: Aggiungi 5mM per 100 ml di Tris-HCl, pH 7,4: 200μl succinato di sodio 1M 80μl methasulfate phenazine (PMS 250mm sciolto in DMSO) 0,25 g NBT Fissare il gel come descritto per il complesso I Complesso III (CIII) Incubare la fetta gel con CIII tampone (fosfato di sodio 50mM, pH 7,2, 0,05% 3,3 &#39;-diaminobenzidina tetraidrocloruro (DAB)) a temperatura ambiente per diverse ore. CIII buffer: Aggiungere per 100 ml di 50 mM di sodio-fosfato, pH 7,2: 50mg diaminobenzidina tetraidrocloruro Fissare il gel come descritto per il complesso I Complesso IV (CIV) Incubare la fetta gel con CIV tampone (fosfato di sodio 50mM, pH 7,2, 0,05% 3,3 &#39;-diaminobenzidina tetraidrocloruro (DAB), 50 micron cavallo cuore citocromo c) a temperatura ambiente per diverse ore. CIV buffer: Aggiungere per 100 ml di 50 mM di sodio-fosfato, pH 7,2: 50mg diaminobenzidina tetraidrocloruro 0.1g cavallo cuore citocromo c Fissare il gel come descritto per il complesso I Complesso V (CV) Incubare la fetta gel con CV buffer (Tris-base da 35 mm, 270 mm glicina, pH 8.3 a 25 ° C, 14 mm MgSO 4, 0,2% w / v Pb (NO 3) 2, 8 mm ATP) a temperatura ambiente per diverse ore. CV buffer: 100 ml di soluzione Per aggiungere: <table border="0"><tbody><tr><td> Tris-base </td><td> 0.424g </td></tr><tr><td> Glicina </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> Non è necessario aggiustare il pH (dovrebbe essere 8.3) - non usare l&#39;acido o di base. Filtrare e aggiungere <table border="0"><tbody><tr><td> ATP </td><td> 0.441g </td></tr></tbody></table> Fissare il gel in metanolo al 50% per 30 min e trasferimento in acqua.

<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>

In questo video, che abbiamo descritto l&#39;estrazione di complessi proteici mitocondriali di cellule staminali embrionali umane, la loro separazione da alte elettroforesi su gel chiaro risoluzione nativa (hrCNE), e la successiva analisi di in-gel saggi di attività catalitica. hrCNE risolve complessi proteici della membrana mitocondriale con una risoluzione paragonabile a quella di blu elettroforesi su gel nativi ed è superiore per in-gel prove di attività. Questa tecnica può essere utilizzato non solo per quantificare respiratoria mit...

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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

I mitocondri sono organelli citoplasmatici che hanno un ruolo primario nel metabolismo cellulare e l&#39;omeostasi, la regolamentazione della rete di segnalazione cellulare, e la morte cellulare programmata. I mitocondri producono ATP, regolare lo stato redox citoplasmatico e Ca2 + equilibrio, catabolizzare acidi grassi, sintesi dell&#39;eme, nucleotidi, ormoni steroidi, amminoacidi, e aiutare assemblare ferro-zolfo in proteine. I mitocondri hanno anche un ruolo essenziale nella produzione di calore. Le mutazioni del genoma mitocondriale causa diversi tipi di disturbo umano. L&#39;accumulo di mutazioni del mtDNA è correlata con l&#39;invecchiamento e si sospetta di avere un ruolo importante nello sviluppo del cancro. Grazie al loro ruolo di vitale importanza in tutti i tipi di cellule, la funzione dei mitocondri devono anche essere critico per le cellule staminali. Progressi fondamentali sono stati fatti nella comprensione della vitalità delle cellule staminali, proliferazione, e la capacità di differenziazione. Ma l&#39;attività funzionale delle cellule staminali, in particolare il loro stato di energia, non era ancora stato studiato in dettaglio. Quasi nulla si sa circa le proprietà mitocondriale di cellule staminali embrionali umane (hESC) e la loro progenie precursore differenziata. Un modo per comprendere e valutare il ruolo dei mitocondri nella funzione hESC e potenziale di sviluppo è quello di misurare direttamente l&#39;attività dei complessi respiratori mitocondriali. Qui, descriviamo alta elettroforesi su gel chiaro risoluzione nativa e la successiva visualizzazione in attività di gel come un metodo per analizzare i cinque complessi della catena respiratoria di hESC.

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

In questo video vi mostreremo come il complesso della catena respiratoria mitocondriale di cellule staminali embrionali umane possono essere analizzati utilizzando nelle prove di attività gel.

Sondaggio per mitocondriale complessa attività di cellule staminali embrionali umane

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 Views.  University of California, Los Angeles. In questo video vi mostreremo come il complesso della catena respiratoria mitocondriale di cellule staminali embrionali umane possono essere analizzati utilizzando nelle prove di attività gel.

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagazione di staminali embrionali umane (ES) Cellule

Ricerca

Biologia

Derivation of Human Embryonic Stem Cells by Immunosurgery

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


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

Derivazione di cellule staminali embrionali umane da Immunosurgery

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

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.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

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.

Etichettatura in vitro di cellule staminali embrionali umane per la Risonanza Magnetica

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.

Cultura e manutenzione di cellule staminali embrionali umane

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.

Preparazione Stendere cromosomiche di cellule staminali embrionali umane per cariotipo

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.

Congelamento e scongelamento cellule staminali embrionali umane

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.

Video Bioinformatica Analisi delle embrionali umane crescita Colony cellule staminali

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.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

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.