This work was supported by a Comprehensive Research Grant from the California Institute for Regenerative Medicine (RC1-00104), a Public Health Service Grant (HL085377) from NHLBI, and a gift from the Pollin Foundation to H.S.B.

The written protocol below is based on published parameters reported by the authors, with some modifications. The protocol is performed with approval by the UCSF Institutional Animal Care and Use (IACUC) and Stem Cell Research Oversight (SCRO) Committees.
I. Culture of human embryonic stem cells (hESCs)
<ol>
 <li>Grow hESCs in standard 6-well (3.5 cm diameter wells) plates.</li>
 <li>At least 12 hours prior to passaging cells, seed mouse embryonic fibroblast (MEF; obtained from CF1 day e12.5 timed-mated mice, irradiated at 1000 Ci) feeder cells onto plates coated with 1% gelatin (6,7). MEFs should be plated at 50-60% confluence, or 4 x 105 cells per 3.5 cm diameter well (Figure 1).</li>
 <li>Passage cells when colonies have reached ~70% confluence (Figure 2).</li>
 <li>To passage the cells, aspirate KSR growth medium (6) from the wells and replace with 0.5 ml medium containing Collagenase IV in KSR at a concentration of 1 mg/ml.</li>
 <li>Incubate plates with Collagenase IV at 37&deg;C/5%CO2 for 5 minutes or until edges of colonies begin to lift from the plate.</li>
 <li>Remove Collagenase IV from the plate, add 0.5 ml of KSR to each well, and manually scrape cells to fully remove from the plate.</li>
 <li>Collect the cell suspension from each well, place in a 15 ml conical tube, and allow cells to settle by gravity for 5 minutes.</li>
 <li>Remove the supernatant from the tube, leaving the cells and medium in a total volume of ~300&mu;l.</li>
 <li>Using a 200 &mu;l micro-pipettor set at full volume, gently pipette the cell suspension 5-6 times to break up the colonies.</li>
 <li>Bring the cell suspension to a total volume of 9 ml per original well of cells using KSR medium (i.e., if one well is harvested, the tube should contain 9 ml). Supplement the medium with recombinant human basic fibroblast growth factor (bFGF) to a concentration of 12.5 ng/ml.</li>
 <li>After washing the new wells containing MEF cells (see step 2) with Dulbecco's phosphate buffered saline (DPBS) to remove all serum, add 3 ml of the cell suspension to each well and incubate the plates at 37&deg;C/5%CO2.</li>
 <li>Change KSR medium with bFGF 24 hours after passage, and every 24-48 hours until cells are ready to be passaged again.</li>
</ol>
II. Preparation of hESC pellet for grafting
<ol>
 <li>At 24 hours before grafting, add ROCK inhibitor to the KSR medium to a final concentration of 10&mu;M to increase cell viability (8). One well of cells at ~70% confluence (5 x 105 cells) will create two pellets, with two pellets inserted per kidney capsule.</li>
 <li>To prepare cell pellets for grafting (9), incubate the ROCK inhibitor-treated cultures with Collagenase IV for 5 minutes at 37&deg;C/5%CO2.</li>
 <li>Aspirate the Collagenase IV from the well and replace with 1 ml DPBS with 10% penicillin/streptomycin (pen/strep).</li>
 <li>Manually scrape the colonies from the plate, rinse the well with an additional 1 ml DPBS with pen/strep, and transfer each 1ml aliquot of cell suspension to a 1.5 ml microfuge tube.</li>
 <li>Spin microfuge tubes briefly at 8,000 x g, aspirate the supernatant, and resuspend the pellets in 500&mu;l DPBS with pen/strep.</li>
 <li>To help bind the cells together for transplant, add 100&mu;l 1 mg/ml Phaseolus vulgaris lectin (PHA) in DPBS to each tube.</li>
 <li>Incubate the cell/PHA suspension at room temperature for 5 minutes, then spin for 2 minutes at 8,000 x g. Rotate the tubes 180&deg; within their rotor wells and spin again for 2 minutes at 8,000 x g.</li>
 <li>Carefully aspirate the supernatant leaving the pellet intact. </li>
 <li>Dislodge the cell pellets from the tubes by gently pipetting 200&mu;l KSR next to the edge of the pellet until it slowly lifts up. </li>
 <li>Once dislodged, immediately transfer the pellets to 0.4&mu;m MILLICELL inserts placed in 3.5 cm diameter wells containing 3 ml KSR, and incubate for 2 hours to overnight at 37&deg;C/5%CO2.</li>
</ol>
III. Renal capsule grafting
<ol>
 <li>Perform all animal procedures in accordance with institutional guidelines using appropriate aseptic technique.</li>
 <li>Anesthetize SCID beige mice (CB17.Cg-PrkdcscidLyst bg/Crl), aged 8-12 weeks, using a combination of inhaled 3% isoflurane/0.5%O2 and intraperitoneal tribromoethanol (Avertin; 25 mg/kg freshly prepared). Two cell pellets per mouse will be engrafted using described techniques (10).</li>
 <li>After placing the mouse on a heating pad to prevent hypothermia, shave the incision area, then disinfect it with chlorhexidine. Make a 2.5 cm incision through the skin just off center but parallel to the spine.</li>
 <li>Keeping the incision area moist with sterile saline, make a smaller incision through the muscle, just below the ribs and ~1 cm from the spine. The incision should be just large enough to pull the kidney through, but small enough that the kidney will not slip back into the abdominal cavity without force.</li>
 <li>Pull the kidney gently through the muscle incision using a glass Pasteur pipette that has been pulled into the shape of a blunt, slightly curved hook.</li>
 <li>Wet the kidney with sterile DPBS to prevent the capsule from drying out. This may be necessary to repeat throughout the procedure, as the capsule is very fragile.</li>
 <li>Using Dumont #5 forceps, gently pull the capsule up from the kidney and make a 2 mm incision using Vannas spring scissors.</li>
 <li>With another glass pipette that has been pulled into a very fine, blunted probe, make a small pocket between the capsule and the kidney.</li>
 <li>Using a large-orifice pipette tip placed on a disposable transfer pipette, take a single cell pellet from the MILLICELL insert. Make sure to take as little extra media along as possible, as excess media can interfere with the pellet transfer.</li>
 <li>While holding up the edge of the incision to create the pocket, gently place the pellet next to the opening and push it into the pocket toward one pole of the kidney with the pulled pipette. </li>
 <li>Repeat the process with a second pellet. Place this under the capsule of the same kidney, but toward the opposite pole.</li>
 <li>Carefully place the capsule back down onto the pellets. The pellets should stay within the pocket, and no sutures are necessary to close the capsule.</li>
 <li>Gently push the kidney back into the abdominal cavity and close the muscle wall using 4-0 silk sutures (or smaller) with attached reverse-cutting needle. The incision should be small enough that only a single suture is necessary.</li>
 <li>Close the skin with a series of 4-5 interrupted stitches, using the same suture material.</li>
 <li>Before placing the mouse back in its cage, administer 0.05 mg/kg buprenorphine (11,12) and 5 mg/kg ketoprofen (12,13) as short-term and long-term analgesics, respectively. Controlled substances should only be used in accordance with institutional guidelines.</li>
 <li>Transfer the animal back to its cage and allow it to recover from anesthesia on a heating pad. This should take about 20-30 minutes.</li>
 <li>Once the mouse is fully ambulatory, it can be returned to the animal housing facility.</li>
</ol>
IV. Teratoma harvest, fixation and sectioning
<ol>
 <li>At 8-12 weeks post-surgery, there should be a barely palpable teratoma near the kidney. Large, fluid-filled cysts will often develop as well, starting at 6-8 weeks. These may necessitate an earlier harvest if they cause distress to the animal. Since teratomas grow at slower rates than cysts, however, you will want to wait as long as possible to obtain a teratoma of sufficient size for analysis (i.e., at least 8 weeks).</li>
 <li>Remove the kidney, teratoma, and any surrounding cysts as a block of tissue from the abdominal cavity using the same surgical approach by which the kidney was originally accessed (see Section III, steps 1-4 above).</li>
 <li>Place the entire block of excised tissue into a tube containing 10% buffered formalin.</li>
 <li>Allow the teratoma to fix overnight at 4&deg;C.</li>
 <li>The following day, remove the teratoma from the tube. Dissect away any cysts and as much kidney as possible without damaging the teratoma itself (Figure 3). Larger teratomas may be cut in half to facilitate dissection and embedding.</li>
 <li>Process the teratoma using a standard paraffin-fixation technique. Automated units are available (e.g., Shandon Hypercenter):
 <table border="0">
 <tbody>
 <tr>
 <td>I.</td>
 <td>80% Flex alcohol</td>
 <td>2 hours</td>
 </tr>
 <tr>
 <td>II.</td>
 <td>80% Flex alcohol</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>III.</td>
 <td>85% Flex alcohol</td>
 <td>1.5 hours</td>
 </tr>
 <tr>
 <td>IV.</td>
 <td>95% Flex alcohol</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>V.</td>
 <td>100% Flex alcohol</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>VI.</td>
 <td>100% Flex alcohol</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>VII.</td>
 <td>100% Flex alcohol</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>VIII.</td>
 <td>Clear-Rite 3</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>IX.</td>
 <td>Clear-Rite 3</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>X.</td>
 <td>Clear-Rite 3</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>XI.</td>
 <td>Paraffin (TissuePrep)</td>
 <td>2 hours</td>
 </tr>
 <tr>
 <td>XII.</td>
 <td>Paraffin (TissuePrep)</td>
 <td>2 hours</td>
 </tr>
 </tbody>
 </table>
 </li>
</ol>
V. Immunohistochemistry and analysis
<ol>
 <li>Once embedded in paraffin, section the teratoma in 5&mu;m serial slices using a rotary microtome, and float sections onto slides.</li>
 <li>Before staining, bake the slides for at least one hour.</li>
 <li>Stain the slides with hematoxylin and eosin using standard methods to identify tissue structures representative of each of the three embryonic germ layers (Figure 4): 
 <table border="0">
 <tbody>
 <tr>
 <td>I.</td>
 <td>Xylene wash</td>
 <td>3-5 minutes</td>
 </tr>
 <tr>
 <td>II.</td>
 <td>Xylene</td>
 <td>3-5 minutes</td>
 </tr>
 <tr>
 <td>III.</td>
 <td>100% alcohol</td>
 <td>3-5 minutes</td>
 </tr>
 <tr>
 <td>IV.</td>
 <td>100% alcohol</td>
 <td>3-5 minutes</td>
 </tr>
 <tr>
 <td>V.</td>
 <td>95% alcohol</td>
 <td>3-5 minutes</td>
 </tr>
 <tr>
 <td>VI.</td>
 <td>80% alcohol</td>
 <td>3-5 minutes</td>
 </tr>
 <tr>
 <td>VII.</td>
 <td>Water wash (several changes)</td>
 <td>2 minutes</td>
 </tr>
 <tr>
 <td>VIII.</td>
 <td>Gill's Hematoxylin #3</td>
 <td>2-5 minutes</td>
 </tr>
 <tr>
 <td>IX.</td>
 <td>Water wash (several changes)</td>
 <td>until water runs clear</td>
 </tr>
 <tr>
 <td>X.</td>
 <td>Scott's water (to blue nuclei)</td>
 <td>3 minutes or more</td>
 </tr>
 <tr>
 <td>XI.</td>
 <td>Water wash (several changes)</td>
 <td>1-2 minutes</td>
 </tr>
 <tr>
 <td>XII.</td>
 <td>Eosin Y</td>
 <td>1 minute</td>
 </tr>
 <tr>
 <td>XIII.</td>
 <td>Water wash (several changes)</td>
 <td>30 seconds</td>
 </tr>
 <tr>
 <td>XIV.</td>
 <td>80% alcohol</td>
 <td>1 minute</td>
 </tr>
 <tr>
 <td>XV.</td>
 <td>95% alcohol</td>
 <td>2 minutes</td>
 </tr>
 <tr>
 <td>XVI.</td>
 <td>95% alcohol</td>
 <td>2 minutes</td>
 </tr>
 <tr>
 <td>XVII.</td>
 <td>100% alcohol</td>
 <td>3 minutes</td>
 </tr>
 <tr>
 <td>XVIII.</td>
 <td>100% alcohol (clean)</td>
 <td>3 minutes</td>
 </tr>
 <tr>
 <td>XIX.</td>
 <td>Xylene</td>
 <td>2 minutes or more</td>
 </tr>
 <tr>
 <td>XX.</td>
 <td>Xylene</td>
 <td>2 minutes or more</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>Mount a coverslip on each slide with Permount.</li>
</ol>
VI. Representative Results
<img src="/files/ftp_upload/2036/2036fig1.jpg" alt="Figure 1" /> Figure 1. Plating of irradiated CF1 mouse embryonic fibroblasts as a feeder layer for culturing undifferentiated hESCs. MEFs are plated at ~50% confluence, as shown here, or 4 x 105 cells per 3.5 cm diameter well in a 6-well culture dish. Bar, 100 &mu;m.
<img src="/files/ftp_upload/2036/2036fig2.jpg" alt="Figure 2" /> Figure 2. Example of subconfluent culture of undifferentiated hESCs on a MEF feeder layer. hESCs are grown on MEF feeder layers until ~70% confluent, as shown here, then passaged as described. Bar, 100 &mu;m.
<img src="/files/ftp_upload/2036/2036fig3.jpg" alt="Figure 3" /> Figure 3. Example of explanted teratomas. Following explantation, the teratoma, kidney, and any accessory tissue are fixed as a block in formalin, then the kidney and accessory tissue are carefully trimmed away before embedding in paraffin.
<img src="/files/ftp_upload/2036/2036fig4.jpg" alt="Figure 4" /> Figure 4. Teratomas derived from undifferentiated hESCs contain tissues representative of all three germ layers in vivo. A teratoma, such as one depicted in Figure 3, was sectioned and stained with hematoxylin and eosin to identify embryonic tissues. hESCs give rise to tissues derived from all three embryonic germ layers (ectoderm (A,B), mesoderm (C), endoderm (D). (A) Nascent neural tube structure. (B) Primitive squamous epithelium. (C) Cartilage surrounded by capsule of condensed mesenchyme. (D) Glandular intestinal structure. Bar, 100 &mu;m. Images reprinted with permission of author.
<img src="/files/ftp_upload/2036/2036fig5.jpg" alt="Figure 5" /> Figure 5. Teratoma analysis can be used to map the fate of tagged, undifferentiated hESCs. As previously published (9), a mixture of specifically tagged hESCs with untagged hESCs can be used to map the fate of the tagged cells in a teratoma formation assay. As shown here, undifferentiated hESCs expressing the surface marker, CD133, and tagged with enhanced green fluorescent protein (eGFP), were mixed with untagged, undifferentiated hESCs. These were used to form teratomas by renal capsule grafting. Immunohistochemical analysis of hematoxylin and eosin-stained teratoma sections with anti-eGFP antibody demonstrates the neuroectodermal fate of the CD133+ hESCs. Teratomas were processed as in Figure 4 and counterstained with anti-eGFP antibody (brown) to localize derivatives of CD133+GFP+ cells within tissues. CD133+-derived cells (arrows) were observed in tissues arising from embryonic ectoderm, specifically neural epithelium (left) and nascent neural tube-like structures (right). Bar, 100 &mu;m. Images reprinted with permission of author.
Gelatin
<ul>
 <li>0.1% Porcine gelatin</li>
 <li>99.9% DPBS
 <ul>
 <li>Add gelatin to DPBS, incubate at 37&deg;C for one hour or until all gelatin goes into solution, and filter sterilize (0.22&mu;m) before use.</li>
 </ul>
 </li>
</ul>
MEF medium
<ul>
 <li>10% Fetal Bovine Serum (Qualified)</li>
 <li>90% Dulbecco's Modified Essential Medium (D-MEM)</li>
 <li>1x L-Glutamine</li>
 <li>1x Pen/Strep
 <ul>
 <li>Filter sterilize before use.</li>
 </ul>
 </li>
</ul>
KSR (Knockout Serum Replacement) medium
<ul>
 <li>20% KnockOut Serum Replacement</li>
 <li>80% KnockOut D-MEM</li>
 <li>1x L-Glutamine</li>
 <li>1x Nonessential Amino Acids</li>
 <li>0.1 mM &beta;-mercaptoethanol/</li>
 <li>12.5 ng/ml bFGF
 <ul>
 <li>Filter sterilize.</li>
 <li>Store at 4&deg;C, but warm to 37&deg;C before adding to cells.</li>
 <li>Add bFGF immediately before use.</li>
 </ul>
 </li>
</ul>
Collagenase IV 
<ul>
 <li>Dissolve 1 mg/ml in KSR medium by placing at 37&deg;C for 1-2 hours or until all powder goes into solution.</li>
 <li>Filter sterilize.</li>
</ul>
Avertin 
<ul>
 <li>0.25 g 2,2,2-tribromoethanol</li>
 <li>0.25 ml 2-methyl-2-butanol
 <ul>
 <li>Incubate at 37&deg;C overnight to form 100% solution.</li>
 <li>Several hours before surgery, dilute to 2.5% working solution using DPBS and incubate at 37&deg;C for at least 1 hour.</li>
 <li>Filter sterilize and aliquot.</li>
 <li>Tribromoethanol is highly light-sensitive, so protect from light at all stages of preparation and use.</li>
 <li>Prepare no more than 12-24 hours before use.</li>
 </ul>
 </li>
</ul>
Buprenorphine
<ul>
 <li>1 mg/ml stock solution in methanol
 <ul>
 <li>Dilute to 0.015 mg/ml working solution using sterile H2O.</li>
 <li>Store at -20&deg;C for up to 1 month.</li>
 </ul>
 </li>
</ul>
Ketoprofen
<ul>
 <li>0.5 mg/ml working solution in DPBS
 <ul>
 <li>Add DPBS, incubate overnight at 37&deg;C for 1-2 hours or until all powder goes into solution.</li>
 <li>Filter sterilize and store at 4&deg;C.</li>
 </ul>
 </li>
</ul>

<ol>
	<li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Hoof, D. V. a. n., D'Amour, K. A., German, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+insulin-producing+cells+from+human+embryonic+stem+cells.">Derivation of insulin-producing cells from human embryonic stem cells.</a> Stem Cell Res. 3, 73-87 (2009).</li><li>Li, Z., Han, Z., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+embryonic+stem+cell-derived+endothelial+cells+for+vascular+diseases.">Transplantation of human embryonic stem cell-derived endothelial cells for vascular diseases.</a> J Cell Biochem. 106, 194-194 (2009).</li><li>Safinia, N., Minger, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+hepatocytes+from+human+embryonic+stem+cells.">Generation of hepatocytes from human embryonic stem cells.</a> Methods Mol Biol. 481, 169-180 (2009).</li><li>Nury, D., Neri, T., Puceat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells+and+cardiac+cell+fate.">Human embryonic stem cells and cardiac cell fate.</a> J Cell Physiol. 218, 455-459 (2009).</li><li>Cowan, C. A., Klimanskaya, I., McMahon, J., Atienza, J., Witmyer, J., Zucker, J. P., Wang, S., Morton, C. C., McMahon, A. P., Powers, D., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+embryonic+stem-cell+lines+from+human+blastocysts.">Derivation of embryonic stem-cell lines from human blastocysts.</a> N Engl J Med. 350, 1353-1356 (2004).</li><li>Schatten, G., Smith, J., Navara, C., Park, J. H., Pedersen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+embryonic+stem+cells.">Culture of human embryonic stem cells.</a> Nat Methods. 2, 455-463 (2005).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat Biotechnol. 25, 681-686 (2007).</li><li>King, F. W., Ritner, C., Liszewski, W., Kwan, H. C., Pedersen, A., Leavitt, A. D., Bernstein, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subpopulations+of+human+embryonic+stem+cells+with+distinct+tissue-specific+fates+can+be+selected+from+pluripotent+cultures.">Subpopulations of human embryonic stem cells with distinct tissue-specific fates can be selected from pluripotent cultures.</a> Stem Cells Dev. 18, 1441-1450 (2009).</li><li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelio-mesenchymal+interactions+in+primordial+gland+structures+which+become+responsive+to+androgenic+stimulation.">Epithelio-mesenchymal interactions in primordial gland structures which become responsive to androgenic stimulation.</a> Anat Rec. 172, 179-195 (1972).</li><li>Kirsch, J. H., Klaus, J. A., Blizzard, K. K., Hurn, P. D., Murphy, S. 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No conflicts of interest declared.

We have adapted a highly efficient teratoma formation assay for the purpose of mapping the fate of differentiating hESCs in an in vivo environment. 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. The fate of the originally selected hESCs can then be determined by immunohistochemistry (Figure 5), as previously reported (9). This method provides a valuable tool for identifying and generating tissue-specific reagents for cell-based therapy.

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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>2-Methyl-2-butanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>240486</td>
<td>Avertin</td>
</tr>
<tr>
<td>2,2,2-Tribromoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T48402</td>
<td>Avertin</td>
</tr>
<tr>
<td>4-0 silk sutures</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>641G</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>233FB</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B7536</td>
<td>&nbsp;</td>
<tr>
<td>Clear-Rite 3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046-341</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C5138</td>
<td>&nbsp;</td>
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>Dupont #5 forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>500233</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>Eosin Y</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>23-245-658</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (Qualified)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Flex alcohol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046344</td>
<td>&nbsp;</td>
<tr>
<td>Gill&#x2019;s Hematoxylin #3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-050-204</td>
<td>&nbsp;</td>
<tr>
<td>Hemostat, straight</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501241</td>
<td>General surgery</td>
</tr>
<tr>
<td>Iris forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>15914</td>
<td>General surgery</td>
</tr>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>K2012</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10829</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25020-081</td>
<td>&nbsp;</td>
<tr>
<td>MILLICELL inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM01250</td>
<td>&nbsp;</td>
<tr>
<td>Nonessential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>Operating scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501754</td>
<td>General surgery</td>
</tr>
<tr>
<td>Paraffin (TissuePrep)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>8002-74-02</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Permount</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SP15-100</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L1668</td>
<td>&nbsp;</td>
<tr>
<td>Porcine gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G6144</td>
<td>&nbsp;</td>
<tr>
<td>ROCK inhibitor</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>Y-27632</td>
<td>&nbsp;</td>
<tr>
<td>SCID beige mice</td>
<td>&nbsp;</td>
<td>Charles River </td>
<td>250</td>
<td>&nbsp;</td>
<tr>
<td>Scott&#x2019;s Wash</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>6697-32</td>
<td>Ricca Chemicals</td>
</tr>
<tr>
<td>Vannas spring scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>14003</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation at JoVE.com

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

fate-mapping-of-human-embryonic-stem-cells-by-teratoma-formation

Research

JoVE Journal

Biology

17.1K Views.  University of California San Francisco. 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.

Video: Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation 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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

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

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

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

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 ...

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 ...