Human embryonic stem cell studies in the Teitell lab are currently supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank Dr. Carla Koehler (UCLA) and members of the Koehler laboratory for providing advice and insights in these and additional studies.

High Resolution Clear Native Gel Electophoresis (hrCNE) for Mitochondrial Complex In-Gel Activity Assays
Human embryonic stem cells (hESCs) were maintained on a monolayer of &#x02E0;-irradiated or mitomycin C-treated mouse embryonic fibroblasts (MEFs), then switched to growth on Matrigel for 5 days in MEF-conditioned medium using standard conditions. To remove contaminating MEFs, hESCs were re-plated on Matrigel and grown in MEF-conditioned medium for an additional 3 days. Immunofluorescence microscopy for pluripotency markers Oct-4 and SSEA-4 was used to confirm the lack of hESC differentiation.
<ol>
<li>Wash cells with warm 1x PBS, pH 7.4, followed by trypsinization (1ml of 1x trypsin per well in a 6-well plate) for 5 min. </li>
<li>Add an equal volume of fresh Trypsin Inhibitor (1mg/ml in 1x PBS, pH 7.4). Harvest cells and centrifuge at 200g, 5 min, room temperature.</li>
<li>Discard the supernatant and resuspend pelleted cells in 0.8 - 1.7ml (based upon the size of the cell pellet) of ice cold mitochondria isolation buffer (83mM sucrose; 6.6mM imidazole/HCl, pH 7.0) with freshly prepared 1&acute; Protease Inhibitor Cocktail. Incubate on ice for 10 min.</li>
<li>Dounce cells with a homogenizer (~40 - 50 strokes). Stain an aliquot of the dounced cells with trypan blue to check for completeness of homogenization using a light microscope. </li>
<li>Centrifuge at 20,000g for 10 min at 4&deg;C to obtain a crude mitochondrial pellet that also contains nuclei and larger cell fragments. Decant and discard the supernatant and weigh the precipitated crude mitochondrial pellet. The pellet can be stored at -80 &deg;C following instant freezing in liquid nitrogen if this is a stopping point. NOTE: Differential centrifugation or other gradient-based procedures can be used for isolating and collecting a pure mitochondrial membrane fraction. See ref. 4 for details. This alternative approach will yield the mitochondrial complex activity per mg of mitochondrial protein instead of complex activity per mg of cell weight or cell number. The concentration of mitochondrial proteins will need to be determined after purification to make this comparison. Also, a different isolation buffer may be required for this approach. For solubilization of the pure mitochondrial membrane fraction, see the note following step 6 below. </li>
<li>Solubilize the crude mitochondrial pellet on ice by vortexing with ice cold solubilization buffer (50mM NaCl, 50mM imidazole/HCl pH 7.0, 1mM EDTA, 2mM 6-aminohexanoic acid). 6-aminohexanoic acid can be replaced with the 1x Protease Inhibitor Cocktail. Use 35&micro;l of solubilization buffer per 20mg of pellet weight. Add 20&micro;l of 10% w/v digitonin per 20mg of pellet weight and mix by flicking the tube. Incubate for 5-10 min on ice. Dodecyl-b-D-maltoside or triton X-100 can be substituted for digitonin to solubilize the proteins. The choice of detergent and its quantity may affect the formation of supercomplexes or multimeric forms of mitochondrial complexes. For more information, see ref.1. NOTE: The quantity of detergent required to solubilize a pure mitochondrial membrane fraction corresponds to a detergent/protein weight ratio ranging from 2-6g/g. The optimal ratio to solubilize mitochondrial membranes is 2.5g/g for dodecyl-b-D-maltoside, 3g/g for triton X-100, and 6g/g for digitonin. </li>
<li>Centrifuge the solubilized pellet at 100,000g for 15 min at 4&deg;C. Collect the clear supernatant.</li>
<li>Add loading buffer to a final 5% w/v glycerol and 0.01% w/v Ponceau S (use a 50% w/v glycerol, 0.1% w/v Ponceau S loading buffer stock solution).</li>
<li>Load gel wells with a volume that contains 20-40mg of the crude mitochondrial pellet per lane. Use an acrylamide gradient gel (4 -13%) with a 3.5% stacking gel. NOTE: Gel preparation and electrophoresis conditions: For gel preparation, use an acrylamide/bis-acrylamide (AA/bisAA) 3 mix as follows: 48g of acrylamide and 1.5g of bisacrylamide (49.5% T, 3% C. Where T is total concentration of AA and bisAA and C is the percentage of crosslinker (bisAA) to total monomer). Gel buffer (3&acute;): 1.5M 6-aminohexanoic acid; 75mM imidazole, pH 7.0</li>
</ol>

<table style="padding-left: 60px;" border="0">
<tbody>
<tr>
<td>Gradient 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>&nbsp;</td>
<td>0.6 ml</td>
</tr>
<tr>
<td>Gel buffer 3&acute;</td>
<td>8.3 ml</td>
<td>8.3 ml</td>
<td>&nbsp;</td>
<td>2.7 ml</td>
</tr>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>5 g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>H2O</td>
<td>14.7 ml</td>
<td>5 ml</td>
<td>&nbsp;</td>
<td>4.7 ml</td>
</tr>
<tr>
<td>Total volume</td>
<td>25 ml</td>
<td>25 ml</td>
<td>&nbsp;</td>
<td>8 ml</td>
</tr>
<tr>
<td>10% APS</td>
<td>139 &micro;l</td>
<td>125 &micro;l</td>
<td>&nbsp;</td>
<td>67 &micro;l</td>
</tr>
<tr>
<td>TEMED</td>
<td>14 &micro;l</td>
<td>12.5 &micro;l</td>
<td>&nbsp;</td>
<td>6.7 &micro;l</td>
</tr>
</tbody>
</table>

Anode buffer: 25mM imidazole/HCl, pH 7.0 Cathode buffer: 50mM Tricine, 7.5mM imidazole/HCl, 0.02% w/v dodecyl-b-D-maltoside, 0.05% w/v deoxycholate, pH 7.0 Pour and run gels in a cold room (4-7 &deg;C). The initial voltage used is 100V. When the sample has entered the stacking gel, the voltage is raised to 500V with the current limited to 15mA (for 0.15 &acute; 14 &acute; 14-cm gels). Electrophoresis is stopped after 3-4 h when the sharp line of the red Ponceau S dye approaches the gel front. Alternatively, a gel can be run overnight with constant voltage at 70-100V.
<ol>
<li>In-gel activity assays. The time of incubation for each assay depends on the efficiency of protein solubilization and the amount of material loaded per well. Complexes I, IV and V usually yield stronger signals than complexes II and III.</li>
</ol>
Complex I (CI)
Incubate the gel slice with CI buffer (5mM Tris-HCl, pH 7.4; 0.1mg/ml NADH; 2.5mg/ml NBT) at room temperature for several hours.
CI buffer: Add per 100ml of 5mM Tris-HCl, pH 7.4:
0.01g NADH
0.25g NBT
Fix the gel in 50% methanol and 10% acetic acid for 15-45 min. The fixed gel is preserved in 10% acetic acid.
Complex II (CII)
Incubate the gel slice with CII buffer (20mM sodium succinate; 0.2mM phenazine methasulfate; 2.5mg/ml NBT; 5mM Tris-HCl pH 7.4) at room temperature for several hours.
CII buffer: Add per 100ml of 5mM Tris-HCl, pH 7.4:
200&micro;l 1M sodium succinate
80&micro;l phenazine methasulfate (250mM PMS dissolved in DMSO)
0.25g NBT
Fix the gel as described for Complex I
Complex III (CIII)
Incubate the gel slice with CIII buffer (50mM sodium phosphate, pH 7.2; 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB)) at room temperature for several hours.
CIII buffer: Add per 100ml of 50mM sodium-phosphate, pH 7.2:
50mg diaminobenzidine tetrahydrochloride
Fix the gel as described for Complex I
Complex IV (CIV)
Incubate the gel slice with CIV buffer (50mM sodium phosphate, pH 7.2; 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB), 50&micro;M horse heart cytochrome c) at room temperature for several hours.
CIV buffer: Add per 100ml of 50mM sodium-phosphate, pH 7.2:
50mg diaminobenzidine tetrahydrochloride
0.1g horse heart cytochrome c
Fix the gel as described for Complex I
Complex V (CV)
Incubate the gel slice with CV buffer (35mM Tris-base, 270mM glycine, pH 8.3 at 25&deg;C, 14mM MgSO4, 0.2% w/v Pb(NO3)2, 8mM ATP) at room temperature for several hours.
CV buffer: Per 100ml of solution add:

<table border="0">
<tbody>
<tr>
<td>Tris-base</td>
<td>0.424g</td>
</tr>
<tr>
<td>Glycine</td>
<td>2.026g</td>
</tr>
<tr>
<td>MgSO4</td>
<td>0.345g</td>
</tr>
<tr>
<td>Pb(NO3)2</td>
<td>0.29g</td>
</tr>
</tbody>
</table>

No need to adjust pH (should be 8.3) - do not use acid or base. Filter and add

<table border="0">
<tbody>
<tr>
<td>ATP</td>
<td>0.441g</td>
</tr>
</tbody>
</table>

Fix the gel in 50% methanol for 30 min and transfer to water.

<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 this video, we have described the extraction of mitochondrial protein complexes from human embryonic stem cells, their separation by high resolution clear native gel electrophoresis (hrCNE), and the subsequent analysis by in-gel catalytic activity assays. hrCNE resolves mitochondrial membrane protein complexes at a resolution comparable to that of blue native gel electrophoresis and is superior for in-gel activity assays. This technique can be employed not only to quantify mitochondrial respiratory complexes I-V but a...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

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.

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 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.