This manuscript is dedicated to the memory of Dr. Wenlan Wang who passed away on May 26, 2011. We thank Dr Douglas A. Kerr for generously providing the HBG3 mES cells used in this study. This work was funded by Nemours, a grant (2 RR016472-10) under the INBRE program of the National Center for Research Resources (NCRR), and a COBRE grant award from the NCRR (5 P20 RR020173-05) to support the Center for Pediatric Research at the Alfred I. duPont Hospital for Children, Wilmington, Delaware, USA.

1. Step 1: Mouse Embryonic Stem (mES) Cell Culture (Timing: 3 days)
1. Plating primary embryonic mouse fibroblasts (PMEF)
<ol>
	<li>Coat four 100-mm tissue culture dishes with gelatin for PMEF adhesion. Add 8 ml of 0.1% gelatin solution (StemCell Technologies) to each dish and incubate for 30 min at room temperature.</li>
	<li>Remove excess gelatin from dishes. Do not rinse the dishes.</li>
	<li>Dilute one vial of Mitomycin C-inactivated PMEF (Millipore) that contains approx. 5 x 106 cells into 40 ml of PMEF media (see recipe in Table 1) and plate onto four 100-mm tissue culture dishes. The PMEF provide a feeder layer for mES cells.</li>
	<li>PMEF should be plated at least one day before adding mES cell cultures. PMEF can be used up to 1 week after plating.</li>
</ol>
2. Plating mES Cells
To monitor the efficiency of mES cell differentiation into motor neurons, we used HBG3, an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9.
<ol>
	<li>Defrost 1 vial of HBG3 mES cells (approx. 1 x 107, contributed by Dr. Douglas Kerr, Johns Hopkins University) in a 37 &deg;C water bath. Add 5 ml of ES media (see recipe in Table 1) in a 15 ml tube and spin down cells at 800 rpm for 5 min.</li>
	<li>Aspirate supernatant and resuspend mES cells in 20 ml of ES media with freshly added Leukemia inhibitory factor (LIF) at a final concentration of 10 ng/ml. NOTE: LIF should be added on the day of use and is required to maintain the mES cells in an undifferentiated state.</li>
	<li>Remove 10 ml PMEF media from the PMEF in each dish and add 10 ml of the HBG3 ES cell suspension to each dish.</li>
	<li>24 h after plating, mES cells form small colonies, similar in size to the colony indicated by the arrowhead in Figure 1B. 72 h after plating, colonies increase in size, many being approximately 100 &mu;m in diameter (Figure 1B, indicated by arrows). These cells are ready for differentiation. Media should be replaced daily to maintain proliferation of cells.</li>
</ol>
2. Step 2: Neural Induction (Timing: 2 days)
To induce differentiation of mES cells into motor neurons, mES cells need to be separated from PMEF and cultivated in a suspension environment. Gelatin coated flasks are used to separate PMEF from mES cells.
<ol>
	<li>When mES colonies are ready for neural induction (defined as differentiation day 0), coat 2 T150 flasks with 0.1% gelatin (18 ml/flask) for 30 min at room temperature and then remove excess gelatin and wash with 1x PBS three times. NOTE: Each 100-mm dish culture will need one T150-gelatin coated flask to separate PMEF.</li>
	<li>Carefully remove media by aspiration, making sure not to dislodge loosely attached colonies. Add 10 ml PBS briefly and then remove by aspiration to complete wash step.</li>
	<li>To separate mES cells from PMEF, add 0.25% trypsin/EDTA (3 ml/dish, StemCell Technologies) and incubate for 3-5 min at 37 &deg;C until the stem cells and PMEF detach from the plate. Add 15 ml fresh ES media without LIF and pipet cells up and down to break colonies (about 15-20 times). Then, add to a 0.1% gelatin-coated T150 flask and incubate for 30 min at 37 &deg;C. After incubation, PMEF should attach to the gelatinized surface while ES cells should be floating. Collect the floating mES cells into a 50 ml tube.</li>
	<li>Spin down at 800 rpm and resuspend cells in 10 ml of Neural Differentiation Medium (see recipe in Table 1).</li>
	<li>Count the cells using a hemocytometer and then plate approximately 2 x 106 mES cells onto a 100-mm bacterial or suspension culture dish. These plates permit growth of suspension cultures and promote formation of embryoid bodies (EBs).</li>
	<li>On differentiation day 1, mES cells form small floating aggregates (EBs) that are visible under a light microscope. Any carryover PMEF attach to the dish. Transfer the suspension cells and media into a 15 ml tube and centrifuge at low speed (500 rpm) for 3 min. Aspirate media carefully, add 10 ml of fresh Neural Differentiation Medium to the pelleted EBs, and then plate cells in a new bacterial dish. In addition to replenishing the media, this step removes any PMEF that carry over from the prior step.</li>
	<li>On differentiation day 2, EBs are ready to be induced into motor neurons.</li>
</ol>
3. Step 3: Motor Neuron Specification (Timing: 5 days)
<ol>
	<li>On differentiation day 2, swirl the culture dish and transfer the media and EBs into a 15 ml tube. Let EBs settle by gravity (~ 10 min) or by centrifugation at low speed (500 rpm) for 3 min.</li>
	<li>Aspirate medium carefully and resuspend EBs in 10 ml of Motor Neuron Differentiation (MND) medium (see recipe in Table 1). Plate EBs in a new bacterial dish and change the media daily for 5 days.</li>
	<li>On differentiation day 7, EBs should be approximately 150 - 200 &mu;m in diameter and should express strong GFP signals (Figure 1C). These EBs are ready to be dissociated and plated onto poly-DL-ornithine/laminin coated dishes or coverslips.</li>
</ol>
4. Step 4: Preparation of Poly-DL-ornithine/laminin Coated Coverslips (Timing: 2 days)
Two days before dissociating EBs (i.e. on differentiation day 5), prepare poly-DL-ornithine/laminin-coated coverslips.
<ol>
	<li>Place 12-mm round coverslips (Warner Instruments) on the bottom of a 24-well plate. Dissolve 25 mg poly-DL-ornithine (Sigma-Aldrich) in 25 ml sterile, double distilled water (d2H2O) to obtain a 10X stock solution (1 mg/ml). When ready to use, make a 1/10 dilution of poly-DL-ornithine with d2H2O to obtain concentration of 100 &mu;g/ml. Add 0.5 ml to each well of the 24-well plate and incubate at 4 &deg;C for overnight.</li>
	<li>The following day (differentiation day 6), aspirate poly-DL-ornithine and let the plates and cover slips air dry in a tissue culture hood. Rinse the plate wells with d2H2O three times. Let air dry for another hour.</li>
	<li>Dissolve 1 mg mouse laminin (Millipore) in 5 ml of iced-cold 1X PBS to make 100x stock solution (200 &mu;g/ml). When ready to use, make 1/100 dilution in iced-cold 1X PBS (2 &mu;g laminin/ml). Add 0.5 ml/well in a 24-well plate and incubate in 4 &deg;C for overnight. Before seeding cells, excess laminin should be removed and wells rinsed with 1X PBS two times. Coverslips can be stored in MND medium (0.5 ml/well).</li>
</ol>
5. Step 5: Axon Elongation (Timing: 2 days)
<ol>
	<li>On differentiation day 7, collect EBs into a 15 ml tube and allow EBs to settle (approximately 3 min). Aspirate media and re-suspend EBs in PBS. Allow EBs to settle and then aspirate PBS. Repeat this step 3 times. Add 1 ml of Accumax (Millipore) to the EBs, gently mix and incubate for 5 min at room temperature. Then aspirate excess Accumax covering the EBs.</li>
	<li>To dissociate EBs, add 3 ml of MND medium. Pipet up and down 20 times using a 1 ml Eppendorf micropipette (blue tip) to disrupt EBs. Incubate for 2 min at room temperature. Transfer cell suspension to a 12x75 mm tube with cell strainer cap (BD Falcon) to obtain a single cell suspension. Repeat this dissociation step with remaining clumps and cells retained by the filter so as to enrich the yield of single cells. The final single cell suspension will be 6 ml.</li>
	<li>Gently mix this single cell suspension and count cells using a hemocytometer. Dilute cells into MND medium (supplemented with 10 ng/ml of each BDNF, GDNF, CNTF, and NT-3) to a density of 2x105 cells per ml. Add cell suspension (0.5 ml/well) to 24-well plates containing poly-DL-ornithine/laminin coated coverslips (previously prepared as described above). The differentiated cells extend long neurites after 2 days in culture (Figure 1D).</li>
</ol>
6. Representative Results
Figure 1A shows an outline of the protocol. In step 1, mES cells are cultivated on PMEF and supplemented with LIF to prevent spontaneous differentiation. In general, undifferentiated mES colonies are round and compact, and have clearly defined edges. The colonies are not in contact with one another. Typically, mES cells should be split at a 1:4 ratio every 2~3 days. However, each individual cell line will grow at a different rate and the split ratio must be determined empirically. Arrows in Figure 1B indicate the typical appearance of mES cells after culturing on a PMEF feeder layer for 3 days. These colonies are large (50-100 &mu;m in diameter) but still maintain round and defined edges. At this stage the colonies are ready for differentiation or subculture. An arrowhead in Figure 1B shows a small mES colony that you typically find 24 h after plating. Four days after plating, mES cells become overgrown. They show a flattened appearance and loss of defined boundaries, indicating that they are beginning to differentiate. Such cells are not optimal for differentiation into neuronal cells.
To differentiate mES cells into motor neurons, mES cells need to be cultivated in suspension conditions to allow EB formation. In step 2, ES cells are transferred onto a culture surface without a feeder layer to promote EB formation. In this step, they are incubated in Neural Differentiation medium supplemented with Noggin, bFGF, and FGF-8 for 2 days to direct cells to a neural lineage. Small EBs can be observed under the microscope at day 1 of differentiation. They should be floating in the medium. Carryover PMEF can also be found at day 1 of differentiation and must be removed. These cells adhere to bacterial dishes so are eliminated when EBs are passaged to a new bacterial dish. Thus, by day 2 of differentiation, few or no PMEF should be seen in the culture dish. In step 3, continued transfer of EBs to new dishes should ensure removal of any remaining PMEF. EBs are cultured in Motor Neuron Differentiation (MND) media supplemented with RA and SAG for 5 days to differentiate the cells into motor neurons. During culture, EBs continue to grow in size and can be seen by the naked eye at differentiation day 3~4. Figure 1C shows the microscopic appearance of EBs on differentiation day 7. Unlike the undifferentiated cells, EBs show strong fluorescence due to expression of GFP. These EBs are optimally differentiated and are ready for dissociation. Flow cytometry showed that 51% &plusmn; 0.8% of the cells from the dissociated EBs had differentiated into GFP expressing cells5. In step 5, culture of these motor neurons in the presence of GDNF, CNTF, BDNF, and NT3 results in extension of long axonal projections. Figure 1D shows the appearance of differentiated cells 2 days after plating of dissociated EBs. Note long neurites extending from the cell bodies of the plated cells. Immunofluorescent staining shows that the GFP+ cells express the pan-neuronal marker (neurofilament-medium chain) and two motor neuron specific markers, (Islet-1 and choline acetyltransferase, Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/3813/3813fig1.jpg" /> 
Figure 1. Differentiation of mES cells into motor neurons (picture modified from Wu et al.5). A) Scheme of the differentiation protocol from mES cells to motor neurons. B) Undifferentiated mES cells form round colonies on the top of a fibroblast feeder layer. They have weak GFP expression. C) mES cells were separated from feeder layers and cultured on low-attachment dishes to form EBs. During the first two days of the differentiation process, cells were exposed to 50 ng/ml Noggin, 20 ng/ml bFGF, and 20 ng/ml FGF-8. Subsequently, they were induced to differentiate with 1 &mu;M retinoic acid and 1 &mu;M sonic hedgehog agonist SAG for 5 days. Differentiated mES cells expressed strong GFP fluorescence. D) After 7-day differentiation, EBs were dissociated and plated on poly-DL-ornithine/laminin coated plates. The differentiated cells extended long neural processes after 2 days in culture. Scale bar = 200 &mu;m. MN = motor neuron. B, C and D are bright field images and B', C' and D' are corresponding fluorescence images.
<img alt="Figure 2" src="/files/ftp_upload/3813/3813fig2.jpg" /> 
Figure 2. Characterization of mES cell-derived motor neurons. Immunofluorescence staining for neurofilament medium chain (NF-M, red), ChAT (red), and Islet-1 (red) was coincident with GFP (green) expression. DAPI (blue) was used to identify the nuclei. Scale bar = 50 &mu;m.

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	<li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110, 385-397 (2002).</li><li>Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+motoneurons+derived+from+mouse+embryonic+stem+cells.">Functional properties of motoneurons derived from mouse embryonic stem cells.</a> J. Neurosci. 24, 7848-7858 (2004).</li><li>Wichterle, H., Peljto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+to+spinal+motor+neurons.">Differentiation of mouse embryonic stem cells to spinal motor neurons.</a> Curr. Protoc. Stem Cell. Biol. Chapter 1, Unit 1H.1.1-Unit 1H.1.9 (2008).</li><li>Kiris, E. <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-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem Cell Res. 6, 195-205 (2011).</li><li>Wu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+assessment+of+a+cell+model+of+spinal+muscular+atrophy.">Proteomic assessment of a cell model of spinal muscular atrophy.</a> BMC Neurosci. 12, 25 (2011).</li><li>McMahon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin-mediated+antagonism+of+BMP+signaling+is+required+for+growth+and+patterning+of+the+neural+tube+and+somite.">Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite.</a> Genes Dev. 12, 1438-1452 (1998).</li><li>Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+posterior+neural+tissue+is+induced+by+FGF+in+the+chick+embryo.">Early posterior neural tissue is induced by FGF in the chick embryo.</a> Development. 125, 473-484 (1998).</li><li>Launay, C., Fromentoux, V., Shi, D. L., Boucaut, 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=A+truncated+FGF+receptor+blocks+neural+induction+by+endogenous+Xenopus+inducers.">A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.</a> Development. 122, 869-880 (1996).</li><li>Sinha, S., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purmorphamine+activates+the+Hedgehog+pathway+by+targeting+Smoothened.">Purmorphamine activates the Hedgehog pathway by targeting Smoothened.</a> Nat. Chem. Biol. 2, 29-30 (2006).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 14071-14076 (2002).</li><li>Lee, S. M., Danielian, P. S., Fritzsch, B., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+FGF8+signaling+from+the+midbrain-hindbrain+junction+regulates+growth+and+polarity+in+the+developing+midbrain.">Evidence that FGF8 signaling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.</a> Development. 124, 959-969 (1997).</li></ol>

No conflicts of interest declared.

The quality of mES cells is the most critical parameter for efficient generation of motor neurons. mES cells must be cultivated on PMEF to prevent spontaneous differentiation. Addition of LIF helps maintain the mES cells in an undifferentiated state. As mES cells divide every 12-15 h, the culture medium becomes depleted rapidly and must be replaced daily.
Efficient activation of the Shh pathway is a critical parameter needed to induce motor neuron specification. Shh protein (R&amp;D System),...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Final concentration </td>
 </tr>
 <tr>
 <td>PMEF </td>
 <td>Millipore</td>
 <td>PMEF-H</td>
 <td>N.A.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PMEF medium</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11965-118</td>
 <td>N.A.</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Invitrogen</td>
 <td>16140-071</td>
 <td>10%</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Invitrogen</td>
 <td>25030-081</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin</td>
 <td>Invitrogen</td>
 <td>15240-063</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>mES medium</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>StemCell Technologies</td>
 <td>36250</td>
 <td>N.A.</td>
 </tr>
 <tr>
 <td>ES Cell-Qualified FBS</td>
 <td>Invitrogen</td>
 <td>16141-079</td>
 <td>15%</td>
 </tr>
 <tr>
 <td>GlutaMax-I</td>
 <td>Invitrogen</td>
 <td>35050-061</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>Non-essential amino acids</td>
 <td>Invitrogen</td>
 <td>11140-050</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>Nucleosides</td>
 <td>Millipore</td>
 <td>ES-008-D</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>&beta;-mercaptoethanol</td>
 <td>Millipore</td>
 <td>ES-007-E</td>
 <td>0.1 mM</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin</td>
 <td>Millipore</td>
 <td>TMS-AB2-C</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>LIF</td>
 <td>Millipore</td>
 <td>LIF2010</td>
 <td>10 ng/ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Neural Differentiation medium</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>StemCell Technologies</td>
 <td>36250</td>
 <td>N.A.</td>
 </tr>
 <tr>
 <td>ES Cult FBS</td>
 <td>StemCell Technologies</td>
 <td>06905</td>
 <td>15%</td>
 </tr>
 <tr>
 <td>Non-essential amino acids</td>
 <td>Invitrogen</td>
 <td>11140-050</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>Mono-thio glycerol</td>
 <td>Sigma-Aldrich</td>
 <td>M-6145</td>
 <td>1mM</td>
 </tr>
 <tr>
 <td>Noggin</td>
 <td>Invitrogen</td>
 <td>PHC1506</td>
 <td>50 ng/ml</td>
 </tr>
 <tr>
 <td>FGF-8</td>
 <td>Invitrogen</td>
 <td>PHG0274</td>
 <td>20 ng/ml</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>Invitrogen</td>
 <td>PHG0024</td>
 <td>20 ng/ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MND medium (differentiation)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ES-Cult Basal Medium-A</td>
 <td>StemCell Technologies </td>
 <td>5801</td>
 <td>N.A.</td>
 </tr>
 <tr>
 <td>Knockout serum replacement</td>
 <td>Invitrogen</td>
 <td>10828-028</td>
 <td>10%</td>
 </tr>
 <tr>
 <td>N-2 supplement</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>ITS Supplement-B</td>
 <td>StemCell Technologies </td>
 <td>07155</td>
 <td>1% </td>
 </tr>
 <tr>
 <td>Ascorbic acid</td>
 <td>StemCell Technologies</td>
 <td>07157</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin</td>
 <td>Millipore</td>
 <td>TMS-AB2-C</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>GlutaMax-I</td>
 <td>Invitrogen</td>
 <td>35050-061</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>D-glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G-8270</td>
 <td>0.15% in d2H2O</td>
 </tr>
 <tr>
 <td>Fibronectin</td>
 <td>StemCell Technologies </td>
 <td>07159</td>
 <td>5 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Sigma-Aldrich</td>
 <td>H3149</td>
 <td>20 &mu;g/ml in d2H2O</td>
 </tr>
 <tr>
 <td>&beta;-mercaptoethanol</td>
 <td>Millipore</td>
 <td>ES-007-E</td>
 <td>0.1 mM</td>
 </tr>
 <tr>
 <td>Retinoic Acid</td>
 <td>Sigma-Aldrich</td>
 <td>R-2625</td>
 <td>1 &mu;M</td>
 </tr>
 <tr>
 <td>SAG</td>
 <td>EMD Chemicals</td>
 <td>566660</td>
 <td>1 &mu;M</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MND medium (Motor Neuron culture)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ES-Cult Basal Medium-A</td>
 <td>StemCell Technologies </td>
 <td>5801</td>
 <td>N.A.</td>
 </tr>
 <tr>
 <td>Knockout serum replacement</td>
 <td>Invitrogen</td>
 <td>10828-028</td>
 <td>10%</td>
 </tr>
 <tr>
 <td>N-2 supplement</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>ITS Supplement-B</td>
 <td>StemCell Technologies </td>
 <td>07155</td>
 <td>1% </td>
 </tr>
 <tr>
 <td>Ascorbic acid</td>
 <td>StemCell Technologies</td>
 <td>07157</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin</td>
 <td>Millipore</td>
 <td>TMS-AB2-C</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>GlutaMax-I</td>
 <td>Invitrogen</td>
 <td>35050-061</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>D-glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G-8270</td>
 <td>0.15% in d2H2O</td>
 </tr>
 <tr>
 <td>Fibronectin</td>
 <td>StemCell Technologies </td>
 <td>07159</td>
 <td>5 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Sigma-Aldrich</td>
 <td>H3149</td>
 <td>20 &mu;g/ml in d2H2O</td>
 </tr>
 <tr>
 <td>&beta;-mercaptoethanol</td>
 <td>Millipore</td>
 <td>ES-007-E</td>
 <td>0.1 mM</td>
 </tr>
 <tr>
 <td>BDNF</td>
 <td>R&amp;D Systems</td>
 <td>248-BD-005/CF</td>
 <td>10 ng/ml</td>
 </tr>
 <tr>
 <td>CNTF</td>
 <td>R&amp;D Systems</td>
 <td>257-NY-010/CF</td>
 <td>10 ng/ml</td>
 </tr>
 <tr>
 <td>GDNF</td>
 <td>R&amp;D Systems</td>
 <td>212-GD-010/CF</td>
 <td>10 ng/ml</td>
 </tr>
 <tr>
 <td>NT-3</td>
 <td>R&amp;D Systems</td>
 <td>267-N3-005/CF</td>
 <td>10 ng/ml</td>
 </tr>
 </tbody>
</table>
N.A. = Non-applicable.
Table 1. PMEF and Media for mES cell culture or differentiation.
<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>0.1% Gelatin</td>
 <td>StemCell Technologies</td>
 <td>07903</td>
 <td>N.A.</td>
 </tr>
 <tr>
 <td>Poly-DL-ornithine</td>
 <td>Sigma-Aldrich</td>
 <td>P0421</td>
 <td>0.1 mg/ml in d2H2O</td>
 </tr>
 <tr>
 <td>Mouse laminin</td>
 <td>Millipore</td>
 <td>CC095</td>
 <td>2 &mu;g/ml in PBS</td>
 </tr>
 <tr>
 <td>0.25% Trypsin/EDTA</td>
 <td>StemCell Technologies</td>
 <td>07901</td>
 <td>N.A.</td>
 </tr>
 <tr>
 <td>Accumax</td>
 <td>Millipore</td>
 <td>SCR006</td>
 <td>N.A.</td>
 </tr>
 </tbody>
</table>
N.A. = Non-applicable.
Table 2. Reagents for coating and dissociation.

efficient differentiation of mouse embryonic stem cells into motor neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

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22.2K visualizações.  Alfred I. duPont Hospital for Children. Desenvolvemos um novo protocolo para melhorar a eficiência de diferenciação in vitro de células-tronco embrionárias em neurônios motores. As células-tronco diferenciadas adquirido neurônios motores apresenta como evidenciado pela expressão de marcadores neuronais e neurônio motor utilizando técnicas de imuno-histoquímica.

Vídeo: Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

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 ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

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

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...