We are grateful to Qing Xu for developing the Nkx2.1::mCherry:Lhx6:GFP dual reporter mESC line as well as Jennifer Tyson, Asif Maroof, and Tim Petros for their early work on helping to develop this protocol. We also thank the CHOP flow cytometry core for technical assistance. This work was supported by an NIH R01 MH066912 (SA) and F30 MH105045-02 (DT).

NOTE: The dual reporter mESC line described in this protocol is available upon request (sande@mail.med.upenn.edu).
1. Media Preparation
NOTE: Warm all media to 37 &#176;C before use in cell culture.
<ol>
	<li>Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
	<ol>
		<li>Add 50 mL fetal bovine serum (FBS) to 449 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), and filter through a 500 mL 0.22 &#181;m pore filter unit.</li>
		<li>Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 &#176;C for up to 1 month or aliquot and store at &#8804; -20 &#176;C.</li>
	</ol>
	</li>
	<li>N2 media (for preparing 500 mL)
	<ol>
		<li>Add 5 mL L-alanine-L-glutamine (100x) and 500 &#181;L 2-Mercaptoethanol (55 mM) to 489.5 mL DMEM: Nutrient Mixture F-12 (DMEM/F-12), and filter through a 500 mL 0.22 &#181;m pore filter unit.</li>
		<li>Add 1 mL antimicrobial agent (50 mg/mL) and 5 mL N2 supplement-B after filtration. Store the media at 4 &#176;C in the dark for up to 1 month.</li>
	</ol>
	</li>
	<li>Serum-free growth medium (KSR) (for preparing 500 mL)
	<ol>
		<li>Add 75 mL serum-free medium supplement, 5 mL L-glutamine (100x), 5 mL MEM Non-Essential Amino Acids (MEM-NEAA) (100x), and 500 &#181;L 2-Mercaptoethanol (55 mM) to 413.5 mL non-glutamine containing DMEM, and filter through a 500 mL 0.22 &#181;m pore filter unit.</li>
		<li>Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 &#176;C for up to 1 month.</li>
	</ol>
	</li>
	<li>Mouse embryonic stem cell media (for preparing 500 mL)
	<ol>
		<li>Add 75 mL stem cell grade FBS, 5 mL MEM-NEAA (100x), 5 mL L-glutamine (100x), and 500 &#181;L 2-Mercaptoethanol (55 mM) to 413.5 mL non-glutamine containing DMEM, and filter through a 500 mL 0.22 &#181;m pore filter unit.</li>
		<li>Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 &#176;C in the dark for up to 1 month or aliquot and store at &#8804; -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Culturing mESCs
<ol>
	<li>Plate mitotically inactive MEF feeder cells in MEF media onto tissue culture treated plates at 3-4 x 104 cells/cm2. Allow at least 12 h for them to settle in a cell culture incubator at 37 &#176;C with &#8805; 95% relative humidity and 5% CO2 before plating the mESCs. If not used immediately, replace the MEF media every 3 days. Dispose of MEFs if not used within 7 days of plating.</li>
	<li>Add mESCs (density range from 1-4 x 104 cells/cm2) to MEF plates in mESC media containing Mouse Leukemia Inhibitory Factor (mLIF) (1,000 U/mL). Incubate the cells at 37 &#176;C with &#8805; 95% relative humidity and 5% CO2.</li>
	<li>Passage mESCs using trypsin-EDTA (0.05% trypsin) (1:5-1:10) once the dish becomes ~ 70-80% confluent or if the colonies are beginning to touch. This typically occurs every 2 days, but can take up to 4 or 5 days depending on the initial plating density.</li>
	<li>Maintain the mESCs on a MEF feeder layer in mESC medium to keep pluripotency.</li>
	<li>Before starting differentiation, passage the cells at least once onto gelatin coated plates without MEFs to dilute out the MEFs.
	<ol>
		<li>Prepare gelatin coated plates by adding 0.1% gelatin in phosphate buffered saline (PBS) with Ca2+/Mg2+ to a tissue culture treated dish and leaving at 37 &#176;C for at least 1 h. Plate 1.5-2 x 106 cells/10 cm plate (2.7-3.6 x 104 cells/cm2) and allow the cells to expand for 2 days before beginning differentiation.</li>
	</ol>
	</li>
</ol>
3. Differentiating mESCs Toward MGE-like Telencephalic Progenitors
<ol>
	<li>Differentiation day (DD) 0 = &#34;float cells&#34;
	<ol>
		<li>After the mESCs have grown on gelatin coated plates for 2 days, aspirate the media and wash the cells once with PBS without Ca2+/Mg2+. Add enough trypsin-EDTA (0.05% trypsin) to cover the surface of the plate (typically 4 mL trypsin-EDTA for one 10 cm tissue culture dish), and place the cells back into the incubator at 37 &#176;C with &#8805;95% relative humidity and 5% CO2 for 4 min.</li>
		<li>After 4 min, quench the trypsin-EDTA using 2 times the volume with mESC media. Transfer the cells to an appropriately sized centrifuge tube and centrifuge the cells at 200 x g for 5 min. After 5 min, remove the tube and aspirate the media without disturbing the pellet. Resuspend the pellet in 1 mL KSR:N2 media (1:1) containing the BMP inhibitor LDN-193189 (250 nM) and the Wnt inhibitor XAV-939 (10 &#181;M).</li>
		<li>Measure the cell concentration using a hemocytometer or automated cell counter. Start growing cells as embryoid bodies (EBs) by adding 75,000 cells/mL in KSR:N2 media (1:1) containing LDN-193189 (250 nM) and XAV-939 (10 &#181;M) in non-adherent tissue culture dishes. Incubate the cells at 37 &#176;C with &#8805; 95% relative humidity and 5% CO2.</li>
	</ol>
	</li>
	<li>On DD1, prepare for cell &#34;landing&#34; by coating tissue culture treated dishes with poly-L-lysine (10 &#181;g/mL in PBS with Ca2+/Mg2+) overnight (O/N) at 37 &#176;C with &#8805; 95% relative humidity or for at least 1 h.</li>
	<li>On DD2, aspirate the poly-L-lysine and coat the plates with laminin (10 &#181;g/mL in PBS with Ca2+/Mg2+) O/N at 37 &#176;C with &#8805; 95% relative humidity. 
	NOTE: While O/N is optimal, as little as 2 h may be sufficient. If plates are not used by the next day, aspirate laminin, replace with PBS without Ca2+/Mg2+, and store at 4 &#176;C for up to 2 weeks.</li>
	<li>DD3 (&#34;land cells&#34;)
	<ol>
		<li>Before beginning EB dissociation, aspirate the laminin and allow the plates to completely dry in a tissue culture hood. Do not use plates that appear shiny or visibly wet. Transfer the EBs with media into a 15 mL tube and centrifuge for 3-4 min at 15 x g or until the EBs have pelleted.</li>
	</ol>
	</li>
	<li>Aspirate the media and add 3 mL of cell detachment solution containing DNase (2 U/mL) and incubate at 37 &#176;C with &#8805; 95% relative humidity and 5% CO2 for 15 min. Gently flick the tube every 3 min to aid in EB dissociation.</li>
	<li>Once the EBs are no longer visible or 15 min have elapsed, add 6 mL KSR:N2 (1:1) containing DNase (1 U/mL) and centrifuge for 5 min at 200 x g.</li>
	<li>Plate the cells in KSR:N2 (1:1) containing LDN-193189 (250 nM), XAV-939 (10 &#181;M), and the ROCK inhibitor Y-27632 (10 &#181;M) at 4.5-5 x 104 cells/cm2.</li>
</ol>
4. SST-enriching Protocol: &#34;DD12 GFP High Sonic Hedgehog (SHH)&#34; (continuation from step 3.7)
<ol>
	<li>On DD5, change media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL), IGF-1 (20 ng/mL), and SSH (50 ng/mL).</li>
	<li>Prepare tissue culture plates for re-plating on day 8 (step 4.4) using the instructions outlined in steps 3.2-3.3.</li>
	<li>On DD7, change media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL), IGF-1 (20 ng/mL), and SHH (50 ng/mL).</li>
	<li>On DD8, re-plate the cells. 
	NOTE: While this step is not essential, re-plating the cells can reduce early-differentiated, unwanted cell types. Re-plating also breaks up balls of cells that make downstream immunohistochemical analyses difficult, and increases the efficiency of FACS at later time points.
	<ol>
		<li>To re-plate cells, detach the cells from the plate with trypsin-EDTA (0.05% trypsin) for 5 min at 37 &#176;C with &#8805;95% relative humidity and 5% CO2. Quench the trypsin-EDTA using 2 times the volume with KSR:N2, and centrifuge at 200 x g for 5 min. Filter the cells through a 40 &#181;m filter tube to remove any clumps. Re-plate the cells at 250,000 cells/cm2 in N2/KSR (1:1) containing FGF-2 (10 ng/mL), IGF-1 (20 ng/mL), and Y-27632 (10 &#181;M) with SHH (50 ng/mL). 
		NOTE: If instead, the decision is made not to re-plate the cells on DD8, change the media with KSR:N2 containing SHH (50 ng/mL) every 2 days from DD7 to DD12 and continue to add IGF-1 (20 ng/mL) and FGF-2 (10 ng/mL) until DD9.</li>
	</ol>
	</li>
	<li>On DD10, change the media with KSR:N2 containing SHH (50 ng/mL).</li>
	<li>On DD12, to obtain cells that are enriched for SST subtypes, use FACS to isolate Lhx6::GFP-only expressing cells.
	<ol>
		<li>To detach the cells, use a non-trypsin containing cell-dissociation reagent rather than trypsin-EDTA. Wash cells once with PBS without Ca2+/Mg2+. Add pre-warmed non-trypsin containing cell-dissociation reagent containing DNase (2 U/mL) to the cells. Place them in the incubator at 37 &#176;C with &#8805;95% relative humidity and 5% CO2 for 10-30 min or until the cells have detached. Gently tap the plates every 5 min to aid the dissociation. 
		NOTE: For DD11-12 cultures, only 10-15 min may be necessary. For DD15-16 cultures, 30 min or more may be required for complete dissociation.</li>
		<li>Once the cells have completely lifted off the plate, proceed to FACS isolation using standard protocols or use the cells in other downstream analyses.</li>
	</ol>
	</li>
</ol>
5. PV-enriching Protocol: &#34;DD11/DD17 mCherry + aPKCi&#34; (continuation from step 3.7)
<ol>
	<li>On DD5, change the media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL) and IGF-1 (20 ng/mL).</li>
	<li>Prepare tissue culture plates for re-plating on DD8 (step 4.4) using the instructions outlined in steps 3.2-3.3.</li>
	<li>On DD7, change the media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL) and IGF-1 (20 ng/mL).</li>
	<li>On DD8, re-plate the cells as described in step 4.4 with the following exceptions: when re-plating the cells, replace SHH with smoothened agonist (SAG; 30 nM) and add atypical PKC inhibitor (aPKCi; 2 &#181;M). Taken together, the re-plating media is now N2/KSR (1:1) containing FGF-2 (10 ng/mL), IGF-1 (20 ng/mL), Y-27632 (10 &#181;M), SAG (30 nM), and aPKCi (2 &#181;M).</li>
	<li>On DD10, change the media with KSR:N2 containing aPKCi (2 &#181;M).</li>
	<li>DD11 
	NOTE: At this point, Nkx2.1::mCherry+ cells are enriched to become PV CIns.
	<ol>
		<li>Detach the cells from the plate for FACS using the same protocol outlined in step 4.6. If the decision is made to collect Nkx2.1::mCherry+ cells at a later differentiation day, disregard this step.</li>
	</ol>
	</li>
	<li>On DD12, change the media with KSR:N2 containing aPKCi (2 &#181;M). On DD14, change the media with KSR:N2 containing aPKCi (2 &#181;M). On DD16, change the media with KSR:N2 containing aPKCi (2 &#181;M). On DD17, collect the Nkx2.1::mCherry+ cells using the same protocol outlined in step 4.6.</li>
</ol>
6. PV-enriching Protocol: &#34;DD17 mCherry Low SHH&#34; (continuation from step 3.7)
<ol>
	<li>On DD5, change the media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL) and IGF-1 (20 ng/mL). On DD7, change the media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL) and IGF-1 (20 ng/mL).</li>
	<li>On DD9, change the media with KSR:N2 (1:1). From this point onward, no additional growth factors are necessary.
	<ol>
		<li>Continue to change the media every 2 days until harvesting the cells on DD17.</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+bias+in+the+mitotic+origins+of+somatostatin-+and+parvalbumin-expressing+interneuron+subgroups+and+the+chandelier+subtype+in+the+medial+ganglionic+eminence.">Spatial and temporal bias in the mitotic origins of somatostatin- and parvalbumin-expressing interneuron subgroups and the chandelier subtype in the medial ganglionic eminence.</a> Cereb Cortex. 22, 820-827 (2012).</li><li>Flames, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delineation+of+multiple+subpallial+progenitor+domains+by+the+combinatorial+expression+of+transcriptional+codes.">Delineation of multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 27, 9682-9695 (2007).</li><li>Fogarty, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+genetic+patterning+of+the+embryonic+neuroepithelium+generates+GABAergic+interneuron+diversity+in+the+adult+cortex.">Spatial genetic patterning of the embryonic neuroepithelium generates GABAergic interneuron diversity in the adult cortex.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 27, 10935-10946 (2007).</li><li>Eiraku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organized+formation+of+polarized+cortical+tissues+from+ESCs+and+its+active+manipulation+by+extrinsic+signals.">Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.</a> Cell Stem Cell. 3, 519-532 (2008).</li><li>Colasante, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Conversion+of+Fibroblasts+into+Functional+Forebrain+GABAergic+Interneurons+by+Direct+Genetic+Reprogramming.">Rapid Conversion of Fibroblasts into Functional Forebrain GABAergic Interneurons by Direct Genetic Reprogramming.</a> Cell Stem Cell. 17, 719-734 (2015).</li><li>Xu, Q., Cobos, I., De La Cruz, E., Rubenstein, J. L., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+cortical+interneuron+subtypes.">Origins of cortical interneuron subtypes.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 24, 2612-2622 (2004).</li><li>Southwell, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneurons+from+embryonic+development+to+cell-based+therapy.">Interneurons from embryonic development to cell-based therapy.</a> Science. 344, 1240622 (2014).</li><li>Tyson, J. A., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+interneuron+transplants+to+study+development+and+treat+disease.">GABAergic interneuron transplants to study development and treat disease.</a> Trends Neurosci. 37, 169-177 (2014).</li><li>Donegan, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+interneuron+transplants+as+a+treatment+for+schizophrenia:+preclinical+validation+in+a+rodent+model.">Stem cell-derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model.</a> Mol Psychiatry. , (2016).</li><li>Deglincerti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+human+embryonic+stem+cells+on+micropatterns.">Self-organization of human embryonic stem cells on micropatterns.</a> Nat Protoc. 11, 2223-2232 (2016).</li><li>Chambers, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nat Biotechnol. 27, 275-280 (2009).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+naive-like+human+pluripotent+stem+cells+based+on+HERVH+expression.">Isolation and cultivation of naive-like human pluripotent stem cells based on HERVH expression.</a> Nat Protoc. 11, 327-346 (2016).</li><li>Zeltner, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+the+biology+of+disease+severity+in+a+PSC-based+model+of+familial+dysautonomia.">Capturing the biology of disease severity in a PSC-based model of familial dysautonomia.</a> Nat Med. 22, 1421-1427 (2016).</li><li>Blahos, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+site+on+the+Galpha+-protein+that+recognizes+heptahelical+receptors.">A novel site on the Galpha -protein that recognizes heptahelical receptors.</a> J Biol Chem. 276, 3262-3269 (2001).</li></ol>

The authors have nothing to disclose.

While this method is highly effective at patterning J1-derived mESCs (SCRC-1010), we have experienced variable success with other mESC lines and clonal isolates. For instance, Foxg1::venus mESCs (EB3-derived; Danjo et al.13) respond poorly to this protocol and Foxg1 induction by DD12 is typically on the order of 1-2%. For reasons that we do not fully understand, another Nkx2.1::mCherry:Lhx6::GFP dual reporter clone (termed JQ59) that was isolated simultaneously as the line described in th...

In both mice and humans, roughly half of all cortical inhibitory interneurons (CIns) originate within a transient subcortical structure known as the medial ganglionic eminence (MGE), where the neuroepithelial progenitors of CIns and other neuronal and glial subgroups express the transcription factor Nkx2.11,2. CIn subgroups or subtypes are defined by intersecting morphological, neurochemical, electrophysiological, and connectivity characteristics3,4. The MGE-derived CIns can be grouped into mostly non-overlapping subgroups based on their expression of either PV or SST, the expression of which correlates with particular electrophysiological and connectivity tendencies5. Dysfunction of interneurons, especially those in the PV subgroup, has been implicated in multiple neuropsychiatric disorders and diseases6,7. The overall goal of this method is to produce stem cell-derived mitotic progenitors and migratory precursors enriched for either PV or SST CIn fate for studying cortical interneuron biology and for use in cell-based therapies.
We have developed a scalable, highly efficient method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mESCs. Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line8, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via FACS and subsequently used in a number of downstream applications. By manipulating a number of signaling pathways, duration of culture, and mode of neurogenesis, we can obtain millions of fluorescently labeled interneuron precursors suitable for a host of downstream applications.
Although several other methods exist for generating MGE-like progenitors from mESCs9,10,11,12,13,14, our method, which relies on the Wnt antagonist XAV-939, is particularly efficient at generating Foxg1/Nkx2.1 co-expressing telencephalic progenitors. In addition, the ability to select for interneuron progenitors or their post-mitotic Lhx6-expressing progeny via our dual reporter system, greatly enhances the capacity to generate distinct progenitors and their progeny.

<table>
	<tbody>
		<tr>
			<td>Bottle-top vacuum filter system</td>
			<td>Corning</td>
			<td>CLS430769</td>
			<td></td>
		</tr>
		<tr>
			<td>Test Tube with Cell Strainer Snap Cap</td>
			<td>ThermoFisher</td>
			<td>Corning 352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts (CF-1 MEF IRR 7M)</td>
			<td>MTI-Globalstem</td>
			<td>GSC-6101G</td>
			<td>1 vial of 7M MEFs is sufficient for four 10-cm TC plates. References: 29,35</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>Ant-pm-2</td>
			<td>Also known as antimicrobial agent. Do not filter with base media -- add after filtration. References: 9,11,36,37</td>
		</tr>
		<tr>
			<td>N2 supplement-B</td>
			<td>Stemcell Technologies</td>
			<td>7156</td>
			<td>Do not filter with base media -- add after filtration</td>
		</tr>
		<tr>
			<td>Glutamax (100x)</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td>Also known as L-alanine-L-glutamine. References: 9,11,38,39</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement (KSR)</td>
			<td>ThermoFisher</td>
			<td>10828028</td>
			<td>Also known as serum-free medium supplement. References: 9,11</td>
		</tr>
		<tr>
			<td>L-glutamine (100x)</td>
			<td>ThermoFisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-NEAA (100x)</td>
			<td>ThermoFisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>ThermoFisher</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut DMEM</td>
			<td>ThermoFisher</td>
			<td>10829018</td>
			<td>Also known as non-glutamine containing DMEM. References: 9,11</td>
		</tr>
		<tr>
			<td>Hyclone FBS</td>
			<td>VWR</td>
			<td>82013-578</td>
			<td>Also known as stem cell grade FBS. References: 9,11</td>
		</tr>
		<tr>
			<td>Tissue culture treated dish (10cm)</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-adherent sterile petri dish (10cm)</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukemia inhibitory factor (mLIF)</td>
			<td>Chemicon</td>
			<td>ESG1107</td>
			<td>Do not freeze, store at 4&#39;C. References: 9,11</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1% Gelatin Solution</td>
			<td>ATCC</td>
			<td>ATCC PCS-999-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P6282</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>ThermoFisher</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ThermoFisher</td>
			<td>A1110501</td>
			<td>Also known as non-trypsin containing cell dissociation reagent. References: 9,11</td>
		</tr>
		<tr>
			<td>RQ1 RNase-Free DNase</td>
			<td>Promega</td>
			<td>M610A</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN-193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>Resuspend in DMSO and store at -80&#39;C in single use aliquots</td>
		</tr>
		<tr>
			<td>XAV939</td>
			<td>Stemgent</td>
			<td>04-0046</td>
			<td>Resuspend in DMSO and store at -80&#39;C in single use aliquots</td>
		</tr>
		<tr>
			<td>rhFGF-2</td>
			<td>R&#38;D Systems</td>
			<td>233-FB</td>
			<td>Resuspend in PBS with 0.1% BSA and store at -80&#39;C in single use aliquots</td>
		</tr>
		<tr>
			<td>rhIGF-2</td>
			<td>R&#38;D Systems</td>
			<td>291-G1</td>
			<td>Resuspend in PBS with 0.1% BSA and store at -80&#39;C in single use aliquots</td>
		</tr>
		<tr>
			<td>ROCK inhibitor (Y-27632)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Resuspend in DMSO and store at -80&#39;C in single use aliquots</td>
		</tr>
		<tr>
			<td>Smoothened agonist (SAG)</td>
			<td>Millipore</td>
			<td>566660-1MG</td>
			<td>Resuspend in H20 and store at -80&#39;C in single use aliquots</td>
		</tr>
		<tr>
			<td>rm Sonic Hedgehog/SHH</td>
			<td>R&#38;D Systems</td>
			<td>464-SH-025</td>
			<td>Resuspend in PBS with 0.1% BSA and store at -80&#39;C in single use aliquots</td>
		</tr>
		<tr>
			<td>PKC&#950; Pseudosubstrate Inhibitor, Myristoylated</td>
			<td>EMD Millipore</td>
			<td>539624</td>
			<td>Resuspend in H20 and store at -80&#39;C in single use aliquots</td>
		</tr>
	</tbody>
</table>

differentiation of mouse embryonic stem cells into cortical interneuron precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

The protocol described in this paper is a modified version of our published protocols15,16,17 and has been optimized for use with our Nkx2.1::mCherry:Lhx6::GFP dual-reporter mESC line. By adding the Wnt inhibitor XAV-939 from DD0-5, combined with re-plating at DD8, we achieve robust Nkx2.1 induction, wherein upwards of 50% of all DAPI+ nuclei in culture are also Nkx2.1 expressing (<strong class="...

Watch this Scientific Journal Video about Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors at JoVE.com

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

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10.3K Views.  University of Pennsylvania. This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Video: Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.