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...

<ol>
	<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>

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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22.1K Views.  Alfred I. duPont Hospital for Children. 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.