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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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 this protocol (termed JQ27), produces less than 1% Nkx2.1-expressing cells when grown under identical conditions. The parent line (J1; ATCC SCRC-1010), however, responds well to this protocol and produces percentages of Nkx2.1-expressing cells on DD12 similar to those shown in Figure 1B. If necessary, the concentrations of XAV-939 and SHH/SAG can be adjusted to optimize the differentiation conditions on a line by line basis. However, our experience using this protocol to differentiate other mESC lines into MGE-like progenitors is limited.
Regarding our Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, not all Nkx2.1+ cells express mCherry. Although nearly all mCherry+ cells express Nkx2.1 protein, reporter expression lags behind Nkx2.1 protein expression, especially during early stages of the differentiation (DD7-9). The fraction of Nkx2.1+ cells that express mCherry steadily increases throughout the course of the differentiation. At peak levels (~ DD12-14) mCherry is expressed in about 30% of all Nkx2.1 expressing progenitors17. Additionally, the same bacterial artificial chromosome used to drive mCherry expression has been used to drive Cre recombinase in Nkx2.1-expression domains of transgenic mice24. In these mice, Cre-expression was weak within Nkx2.1-expressing cells of the dorsal-most region of the MGE, and fate-mapping with this mouse appeared to under-label SST-expressing CIns that are known to be largely generated from this region25,26,27,28. However, despite these discrepancies, millions of GFP or mCherry-expressing cells can be isolated with only a few hours of FACS.
There are two main advantages of this technique. First, when combined with our Nkx2.1::mCherry:Lhx6::GFP dual-reporter mESC line, it is possible to obtain large numbers of interneuron precursors enriched to become PV or SST-fated cortical interneuron subtypes. The second main advantage is the relative ease with which large numbers of interneuron fated cells can be obtained. Although several other modified EB techniques exist to differentiate MGE progenitors and neurons, these protocols typically rely on the use of Wnt-inhibitory molecules such as Dkk113,14,29. We have found that the Wnt inhibitor XAV-939 greatly enhances the generation of pallidal telencephalic progenitors, as evidenced by Foxg1 and Nkx2.1 expression15.
As mentioned earlier, several other methods exist for generating MGE-like progenitors from mESCs. Watanabe et al.14 pioneered the field of telencephalic differentiation from mESCs by being the first to develop a serum-free suspension culture method followed by re-plating onto an adherent surface. They found that the Wnt and Nodal antagonists, Dkk1 and LeftyA, respectively, efficiently drove mESCs into early neuroectodermal lineages13. By applying SHH to these cultures, they observed that between 5-15% of all cells in culture co-expressed Nkx2.1/Foxg1 and could be regarded as MGE-like progenitors13. However, while laying the foundation for many studies to come, this study did not have a way to isolate MGE progenitors or enrich for particular CIn subtypes.
Several years later, Danjo et al.13 used the same serum-free suspension culture method in combination with the timed administration of various growth factors, in particular SHH, to achieve subregional specification of mESC-derived ventral telencephalic tissues. Using a Foxg1::venus reporter line, they found that SHH administration from DD3-12 after Dkk1 treatment resulted in between 45-68% of all cells in culture expressing the Foxg1::venus reporter13. Within the Foxg1::venus+ population, approximately 32% of cells expressed Nkx2.113. By substituting SHH for SAG and adding Fgf8, they further enhanced the fraction of Nkx2.1 progenitors within the Foxg1::venus+ population to approximately 41%13. When assayed for fate, these cells produced approximately 17% SST, 21% PV, 18% NPY, and 8% calretinin subtypes13. However, while this study described a method to specify CGE versus MGE-derived CIn fates, it did not describe a method to selectively enrich for PV or SST subtypes.
The following year, Cambray et al.11 used a serum-free monolayer method with re-plating on DD5 to study the effect of activin on the differentiation and identity of telencephalic neural precursors derived from mouse and human ESCs. Although this study did not report the percentage of Nkx2.1 cells generated in their protocol, they report that approximately 54% of cells in culture co-expressed Nestin/Foxg1 on DD2 (5 days of growth in serum free media followed by an additional 2 days of culture after re-plating)11. They also reported that with the addition of activin, ~55% of cells co-expressed nestin and CouptfII, indicating that these cells may represent caudal ganglionic eminence-like cells, which produce the majority of calretinin-expressing cortical interneurons11. Indeed, by 8 days in culture (13 days overall) 39% of cells were found to co-express calretinin/BIII-tubulin, while 59% of cells were found to co-express GABA/BIII-tubulin11. This study did not examine the production of PV or SST subtypes.
Again using a modified EB method, Chen et al.12 demonstrated that MGE-specific enhancer elements could be used to follow and purify stem cell derived MGE-like cells12. In this study, they compared the effect of different culture conditions during directed differentiations of mESCs into MGE-like progenitors using an Lhx6::GFP reporter line; the same line which served as the parent line for the Nkx2.1::mCherry:Lhx6::GFP dual-reporter line described in this manuscript. They showed that by adding Dkk1 to the culture conditions from DD0-3, followed by the addition of SAG, upwards of ~ 10% of all cells in culture expressed Lhx6::GFP12. They also found that the same protocol, applied to the Foxg1::venus line described by Danjo et al.12, resulted in approximately 37% of all cells expressing the Foxg1::venus reporter. When Lhx6::GFP cells were transplanted in vivo, the following average percentages of CIn subtype markers was observed: 22% PV, 58% SST, and 16% NPY12.
More recently, other groups have used the forced expression of lineage-defining transcription factors to directly specify CIns. Perhaps the first demonstration of this was by Petros et al.10 who used a doxycycline-inducible promoter to drive Nkx2.1 expression in Ainv15 mESCs that were modified to contain an Lhx6::GFP bacterial artificial chromosome. This study used culture conditions similar to those described in the current manuscript but without the addition of XAV-93910. However, for reasons that are poorly understood, a lower percentage of Lhx6::GFP+ cells were observed under the same differentiation conditions compared to when using the J1 Lhx6::GFP parent line described in this manuscript10. Despite achieving similar levels of Foxg1 expression (total % unknown), there was a moderate reduction in the total number of Nkx2.1 cells with no greater than 2% of all cells expressing the Lhx6::GFP reporter10. This study highlights the inherent differences that exist between various ESC lines and the challenges that researchers face when attempting to apply differentiation protocols to ESC lines different from those that were used to originally develop the protocols.
Using an elegant approach, Au et al.9 used the sequential forced expression of transcription factors to generate specific subtypes of cortical interneurons using the same ventral telencephalic differentiation paradigm developed by Watanabe et al.14 While they do not report the exact percentages of Foxg1+, Nkx2.1+, and Olig2+ precursors produced in culture, they state that approximately 50% of all cells in culture differentiated into GABAergic neurons9. At 11 days post-differentiation, EBs were dissociated and transplanted into the MGE of E13.5 host embryos9. A small percentage of these (less than 1%) migrated from the MGE into the cortex, where they were identified by their Dlx5/6-eGFP reporter and analyzed for fate9. The authors observed a variety of percentages of PV, SST, and CGE-like fates based upon their chosen forced induction strategy9. Interestingly, with expression of the transcription factor Lmo3, they observed on average approximately 57% PV, 21% SST, and 15% CGE-derived subtypes (SST-/reelin+ or VIP-only+)9. The greatest percentage of SST subtypes observed was obtained using an Nkx2.1/Dlx2 GOF line, which produced approximately 32% SST, 35% PV and 25% CGE-derived subtypes9.
In 2015, Colasante et al.30 showed that the forced expression of five transcription factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) within fibroblasts from GAD67-GFP reporter mice converted 15% of all cells into GABAergic-like CIns. Remarkably, approximately 90% of these cells went on to express PV, with only rare cells expressing SST30. Although this method was not applied to mESCs, it was used with human iPSCs, where it was estimated that approximately 30% of all cells adopted a GABAergic identity30.
As shown in Figure 2, there are several ways to generate enriched populations of PV or SST CIns. The main advantages of the PV-protocol utilizing aPKCi over the DD17 protocol are the greater number of cells one can isolate, cell viability, and duration of culture. Although we do not show data for DD17 induction levels in Figure 1, there are roughly twice as many mCherry+ cells at DD11 as there are at DD17 (data not shown). When collecting cells for transplantation, we find that a greater number of starting cells (see below) is beneficial, since roughly 30-50% of cells are lost in the process of concentrating them for injection. In addition, a greater number of cells can be obtained with shorter FACS times, which limits the amount of time that the cells are on ice and lowers the overall cost. Probably because a higher percentage of the Cherry+ cells are progenitors, cells from earlier stages of culture (e.g., DD11) exhibit greater survival when compared to DD17 cells after transplantation into neonatal neocortex. When large numbers of viable cells are needed, for example in the case of cell transplantation to treat epilepsy, having both a larger starting population and greater viability is a major advantage.
As depicted in Figure 2, depending on the protocol, approximately 25-39% of Lhx6::GFP+ cells analyzed 30 days post-transplantation do not express detectable levels of PV or SST protein with routine immunohistochemistry. Generally, these PV-/SST- cells express GABA as well as Sox6, a marker of MGE-derived cortical interneurons (see Tyson et al.15, Tischfield et al.17). Less than 1% of PV-/SST- Lhx6::GFP+ cells express markers of other interneuron subgroups, such as calretinin, reelin, or neuropeptide Y (data not shown). This is similar to in vivo fate mapping studies which have shown that between ~ 15-25% of MGE-derived interneurons do not express PV or SST23,25,31. When Nkx2.1::mCherry+ or Lhx6::GFP+ cells are isolated and plated onto rat cortical feeders in vitro, we have found that ~ 40-70% of Lhx6::GFP+ cells analyzed after 28 days in culture are PV-/SST-, with a majority of those that do stain positive expressing SST. This increase in PV-/SST- cells may be due to the absence of an extrinsic maturational signal that is present in vivo, as may especially be the case for mature PV expression, or suboptimal electrophysiological input from feeder cells. Whatever the reason may be, we prefer to transplant cells into neonatal neocortex for fate analysis rather than in vitro differentiation.
For most transplantation experiments, we recommend floating on DD0 a minimum of two 10-cm non-adherent tissue culture dishes each containing 7.5 x 105 mESCs in 10 mL of N2/KSR (7.5 x 104 cells/mL; 20 mL total volume). Together, both plates will typically yield 8-12 x 106 cells after EB dissociation on DD3, of which 5.4 x 106 cells can be used to seed two 6-well tissue culture treated plates (50,000 cells/cm2; approximately 110 cm2 total). By DD8, each 6-well plate yields approximately 5-7 x 107 cells, which can then be used to seed an additional three to five 6-well plates at a density of 250,000 cells/cm2 (requiring a total of 4.05 x 107 cells in the case of three 6-well plates or 6.75 x 107 cells in the case of five 6-well plates). By DD14, each 6-well plate will typically yield 1-2.5 x 106 mCherry or GFP-expressing cells. Depending on the needs of the experiment, we have floated a minimum of 7.5 x 105 mESCs for small-scale immunohistochemistry analyses to upwards of 6 x 106 mESCs for large transplantation projects.
The most critical steps of the protocol are maintaining the mESCs in a pluripotent state prior to beginning differentiation and the landing/dissociation step on DD3. If the cells have been maintained properly and the landing/dissociation on DD3 is done carefully, the differentiation will usually be successful. Only stem cells that appear healthy and should be used for the differentiation. If EBs fail to form between DD0-3 or form multiple, small, asymmetrical bodies, the differentiation is likely to be unsuccessful. Additionally, during EB dissociation on DD3, the EB&#39;s should be carefully monitored until no longer visible by eye to avoid prolonged exposure to the non-trypsin containing cell-dissociation regent.
The results presented in this paper were obtained under optimized conditions using quality-controlled reagents. This is important to note since we occasionally have difficulty achieving robust Nkx2.1 induction, and by extension, mCherry and GFP reporter expression. Based on our experience, we believe that lot-to-lot variability of FBS most likely accounts for these changes. As such, it is wise to test different lots of FBS to determine which work best for maintaining mESCs. Although we have not used the 2i/LIF system (serum-free medium containing the MEK inhibitor PD03259010 and GSK-3&#945;/&#946; inhibitor CHIR99021), it is possible that serum-free mESC conditions may produce more consistent results. We have not, however, tested this hypothesis and have no data as to whether the use of serum-free mESC media affects the differentiation of mESCs into MGE-like progenitors. In addition, be sure to use fresh growth factors when possible by aliquoting them for single use. Depending on the user, cell densities may need to be increased to achieve adequate cell survival, especially during the landing step on DD3.
Interneurons have the remarkable capacity to survive, migrate, and integrate into host- circuitry post-transplantation. As such, this protocol can be used to transplant interneuron precursors into epileptic mouse models and other models of neurological and neuropsychiatric disease (see Southwell et al.32 and Tyson and Anderson33 for reviews of interneuron cell based therapies). For example, in a recent study Donegan et al.34 enriched populations of PV and SST CIns for transplantation into a mouse model of schizophrenia. They found that PV and SST enriched transplants produced different effects on mouse behavior, with PV enriched populations normalizing the schizophrenia endophenotypes that they were studying.
In recent years, the PiggyBac transposon system has been used to achieve stable expression of transgenes in a number of systems. We have used this system to quickly and easily create stably expressing doxycycline-inducible miRNA constructs to study the role of particular genes during interneuron development. We are also using this technology to create dual reporter mESC lines that express channelrhodopsin-2 (ChR2) to optogenetically drive mESC derived interneurons or DREADD receptor technology to turn on or off transplanted cells en masse.

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: &lt;sup class=&quot;xref&quot;&gt;29&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;35&lt;/sup&gt;</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: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;36&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;37&lt;/sup&gt;</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: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;38&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;39&lt;/sup&gt;</td></tr><tr><td>KnockOut Serum Replacement (KSR)</td><td>ThermoFisher</td><td>10828028</td><td>Also known as serum-free medium supplement. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</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: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</td></tr><tr><td>Hyclone FBS</td><td>VWR</td><td>82013-578</td><td>Also known as stem cell grade FBS. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</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&#039;C. References: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</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: &lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;&lt;sup&gt;,&lt;/sup&gt;&lt;sup class=&quot;xref&quot;&gt;11&lt;/sup&gt;</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&#039;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&#039;C in single use aliquots</td></tr><tr><td>rhFGF-2</td><td>R&amp;amp;D Systems</td><td>233-FB</td><td>Resuspend in PBS with 0.1% BSA and store at -80&#039;C in single use aliquots</td></tr><tr><td>rhIGF-2</td><td>R&amp;amp;D Systems</td><td>291-G1</td><td>Resuspend in PBS with 0.1% BSA and store at -80&#039;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&#039;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&#039;C in single use aliquots</td></tr><tr><td>rm Sonic Hedgehog/SHH</td><td>R&amp;amp;D Systems</td><td>464-SH-025</td><td>Resuspend in PBS with 0.1% BSA and store at -80&#039;C in single use aliquots</td></tr><tr><td>PKC&amp;zeta; Pseudosubstrate Inhibitor, Myristoylated</td><td>EMD Millipore</td><td>539624</td><td>Resuspend in H20 and store at -80&#039;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.

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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Neuroscience

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

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Developmental Biology

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

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 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.

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 ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells

Source: Tischfield, D. J., et al <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56358">Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors.</a> J. Vis. Exp. (2017)
This video presents a method for generating cortical interneuron precursors from mouse embryonic stem cells. The protocol entails culturing the stem cells in media containing specific small molecule inhibitors, which facilitate stem cell proliferation and aggregation into embryoid bodies (EBs). Subsequently, the EBs are dissociated with enzymes and plated on culture plate wells coated with adhesion proteins to promote their differentiation into cortical interneuron precursors.

1. Media Preparation
NOTE: Warm all media to 37 &#38;#176;C before use in cell culture.

	Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
	
	...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Source: Mao, X., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Source: Hubbard, K. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52361">Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for embryonic stem cell differentiation into a networked population of neurons with functioning synapses. It outlines the steps involved in embryonic stem cell differentiation into neurons, induction of neuronal network formation, and enrichment of the culture with networked neurons.

1. Neuronal Differentiation
NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented ...