Name: differentiation of mouse embryonic stem cells into cortical interneuron precursors
Uploaded: 2017-12-03T14:00:00
Description: Watch this Scientific Journal Video about Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors at JoVE.com

We zijn dankbaar Qing Xu voor de ontwikkeling van de Nkx2.1::mCherry:Lhx6:GFP dual verslaggever mESC lijn evenals Jennifer Tyson Asif Maroof en Tim Petros voor hun vroege werk op het helpen ontwikkelen van dit protocol. Wij danken ook de CHOP stroom cytometry kern voor technische bijstand. Dit werk werd gesteund door een NIH R01 MH066912 (nv) en de F30 MH105045-02 (DT).

Opmerking: De dubbele verslaggever mESC regel beschreven in dit protocol is beschikbaar op verzoek (sande@mail.med.upenn.edu).
1. Media voorbereiding
Opmerking: Warm alle media tot 37 ° C vóór gebruik in celkweek.
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Muis embryonale Fibroblast (MEF) media (voor het voorbereiden van 500 mL)
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<li>Meng 50 mL foetale runderserum (FBS) 449 mL Dulbecco van bewerkt Eagle's Medium (DMEM), en filter via een 500 mL 0,22 µm porie filter eenhei...

<ol>
	<li>Jones, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+of+cortical+interneurons:+mouse+versus+monkey+and+human.">The origins of cortical interneurons: mouse versus monkey and human.</a> Cereb Cortex. 19, 1953-1956 (2009).</li><li>Wonders, C. P., 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=The+origin+and+specification+of+cortical+interneurons.">The origin and specification of cortical interneurons.</a> Nature reviews. Neuroscience. 7 (6), 687-696 (2006).</li><li>Ascoli, G. 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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=Duration+of+culture+and+sonic+hedgehog+signaling+differentially+specify+PV+versus+SST+cortical+interneuron+fates+from+embryonic+stem+cells.">Duration of culture and sonic hedgehog signaling differentially specify PV versus SST cortical interneuron fates from embryonic stem cells.</a> Development. 142, 1267-1278 (2015).</li><li>Maroof, A. 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=Directed+differentiation+and+functional+maturation+of+cortical+interneurons+from+human+embryonic+stem+cells.">Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells.</a> Cell Stem Cell. 12, 559-572 (2013).</li><li>Tischfield, D. J., Kim, J., 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=Atypical+PKC+and+Notch+Inhibition+Differentially+Modulate+Cortical+Interneuron+Subclass+Fate+from+Embryonic+Stem+Cells.">Atypical PKC and Notch Inhibition Differentially Modulate Cortical Interneuron Subclass Fate from Embryonic Stem Cells.</a> Stem Cell Reports. 8, 1135-1143 (2017).</li><li>Liodis, P., 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=Lhx6+activity+is+required+for+the+normal+migration+and+specification+of+cortical+interneuron+subtypes.">Lhx6 activity is required for the normal migration and specification of cortical interneuron subtypes.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 27, 3078-3089 (2007).</li><li>Du, T., Xu, Q., Ocbina, P. J., 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=NKX2.1+specifies+cortical+interneuron+fate+by+activating+Lhx6.">NKX2.1 specifies cortical interneuron fate by activating Lhx6.</a> Development. 135, 1559-1567 (2008).</li><li>Marin, O., Anderson, S. A., Rubenstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+molecular+specification+of+striatal+interneurons.">Origin and molecular specification of striatal interneurons.</a> Journal of Neuroscience. 20, 6063-6076 (2000).</li><li>Xu, Q., Wonders, C. P., 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=Sonic+hedgehog+maintains+the+identity+of+cortical+interneuron+progenitors+in+the+ventral+telencephalon.">Sonic hedgehog maintains the identity of cortical interneuron progenitors in the ventral telencephalon.</a> Development. 132, 4987-4998 (2005).</li><li>Glickstein, S. B., Alexander, S., Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+cyclin+D2+and+D1+protein+expression+distinguish+forebrain+progenitor+subsets.">Differences in cyclin D2 and D1 protein expression distinguish forebrain progenitor subsets.</a> Cereb Cortex. 17, 632-642 (2007).</li><li>Petros, T. J., Bultje, R. S., Ross, M. E., Fishell, G., 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=Apical+versus+Basal+Neurogenesis+Directs+Cortical+Interneuron+Subclass+Fate.">Apical versus Basal Neurogenesis Directs Cortical Interneuron Subclass Fate.</a> Cell Rep. 13, 1090-1095 (2015).</li><li>Xu, Q., Tam, M., 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=Fate+mapping+Nkx2.1-lineage+cells+in+the+mouse+telencephalon.">Fate mapping Nkx2.1-lineage cells in the mouse telencephalon.</a> J Comp Neurol. 506, 16-29 (2008).</li><li>Wonders, C. P., 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+spatial+bias+for+the+origins+of+interneuron+subgroups+within+the+medial+ganglionic+eminence.">A spatial bias for the origins of interneuron subgroups within the medial ganglionic eminence.</a> Dev Biol. 314, 127-136 (2008).</li><li>Inan, M., Welagen, J., 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=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. 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Hoewel deze methode is zeer effectief bij patronen J1-afgeleide mESCs (SCRC-1010), hebben we ervaren variabele succes met andere mESC lijnen en klonen isolaten. Bijvoorbeeld, Foxg1::venus mESCs (EB3-afgeleid; Danjo et al. 13) reageren slecht bij dit protocol en de Foxg1 inductie door DD12 is meestal over de volgorde van 1-2%. Om redenen die wij niet volledig begrijpen, produceert een andere Nkx2.1::mCherry:Lhx6::GFP dual verslaggever kloon (zogenaamde JQ59) dat werd geïsoleerd gelijktijd...

In zowel mens als muizen, ongeveer de helft van alle corticale remmende interneuronen (CIns) hun oorsprong vinden binnen een voorbijgaande subcorticale structuur die bekend staat als de mediale ganglionaire eminentie (MGE), waar de neuroepithelial progenitorcellen van CIns en andere neuronale en gliale subgroepen express de transcriptiefactor Nkx2.11,2. CIn subgroepen of subtypen worden gedefinieerd door de doorsnede van de morfologische, neurochemical, elektrofysiologische en connectiviteit kenmerken3,4. De CIns MGE-afgeleid kunnen worden gegroepeerd in meestal niet-overlappende subgroepen op basis van hun uitdrukking van PV of SST, de uitdrukking van welke correlaten met name elektrofysiologische en connectiviteit tendensen5. Dysfunctie van interneuronen, vooral die in de PV deelgroep, heeft betrokken bij meerdere neuropsychiatrische stoornissen en ziekten6,7. Het algemene doel van deze methode is mitotische progenitoren stamcel-afgeleide en trekkende precursoren verrijkt voor PV of SST CIn lot voor de studie van biologie van de corticale interneuron en voor gebruik in cel-gebaseerde therapieën te produceren.
We hebben een schaalbare, uiterst efficiënte methode voor de afleiding van de Nkx2.1-uiten interneuron progenitoren en hun nakomelingen van mESCs ontwikkeld. Met behulp van een Nkx2.1::mCherry:Lhx6::GFP dual verslaggever mESC lijn8, Nkx2.1 progenitoren of hun Lhx6-uiten na mitotische nakomelingen kunnen worden geïsoleerd via FACS en vervolgens gebruikt in een aantal downstream toepassingen. Door het manipuleren van een aantal signaalroutes, duur van de cultuur, en de wijze van neurogenese, kunnen we miljoenen fluorescently geëtiketteerde interneuron precursoren geschikt voor tal van downstream toepassingen.
Hoewel er verschillende andere methoden bestaan voor het genereren van MGE-achtige voorlopercellen van mESCs9,10,11,12,13,14, onze werkwijze, die gebaseerd op de Wnt is antagonist XAV-939, is bijzonder efficiënt op het genereren van Foxg1/Nkx2.1 mede uiting van telencephalic voorlopercellen. Daarnaast verbetert de mogelijkheid om te kiezen voor interneuron progenitoren of hun post mitotische Lhx6-uiten nageslacht via ons systeem van de dubbele verslaggever, sterk het vermogen om verschillende progenitoren en hun nakomelingen te genereren.

<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 corticale interneuronen zijn een heterogene populatie van cellen die spelen een kritieke rol in de uitvoer van excitatory piramidale neuronen reguleren, alsmede het synchroniseren van de uitgangen van de piramidale neuron ensembles. Tekorten in interneuron functie zijn betrokken bij allerlei neuropsychiatrische aandoeningen, waaronder schizofrenie, autisme en epilepsie. De afleiding van corticale interneuronen vanaf embryonale stamcellen niet alleen voorziet in de studie van hun ontwikkeling en functie, maar verschaft inzicht in de moleculaire mechanismen die ten grondslag liggen aan de pathogenese van corticale interneuron-gerelateerde aandoeningen. Interneuronen ook de opmerkelijke capaciteit hebben om te overleven, migreren en integreren van host corticale circuits na transplantatie, waardoor ze ideale kandidaten voor gebruik in cel-gebaseerde therapieën. Hier presenteren we een schaalbare, uiterst efficiënte, bewerkt, embryoid lichaam-aan-enkelgelaagde-methode voor de afleiding van de Nkx2.1-uiten interneuron progenitoren en hun nakomelingen van muis embryonale stamcellen (mESCs). De opdrachtregel Nkx2.1::mCherry:Lhx6::GFP dual verslaggever mESC, kunnen Nkx2.1 progenitoren of hun Lhx6-uiten na mitotische nakomelingen worden geïsoleerd via fluorescentie-activated cell sorting (FACS) en vervolgens gebruikt in een aantal downstream toepassingen. We bieden ook methoden om te verrijken voor parvalbumin (PV) of Somatostatine (SST) interneuron subgroepen, die nuttig kan zijn voor het bestuderen van aspecten van lot bepaling of voor gebruik in therapeutische toepassingen die van interneuron deelgroep profiteren zouden-verrijkt transplantatiecentra.

Het protocol beschreven in dit document is een gewijzigde versie van onze gepubliceerde protocollen15,16,17 en is geoptimaliseerd voor gebruik met onze Nkx2.1::mCherry:Lhx6::GFP dual-reporter mESC lijn. Door het toevoegen van de Wnt-remmer XAV-939 van DD0-5, gecombineerd met het opnieuw plating op DD8, bereiken wij robuuste Nkx2.1 inductie, waarin meer dan 50% van alle DAPI + kernen in cultuur oo...

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

Dit protocol wordt een methode voor het genereren van corticale interneuron progenitoren en post mitotische interneuron precursoren van muis embryonale stamcellen met een gewijzigde embryoid lichaam-aan-enkelgelaagde methode beschreven. Deze progenitoren/precursoren kunnen worden gebruikt in vitro fluorescently gesorteerd en getransplanteerd in neonatale neocortex voor het bestuderen van de bepaling van het lot of gebruikt in therapeutische toepassingen.

Differentiatie van muis embryonale stamcellen in de corticale Interneuron precursoren

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

The overall goal of this procedure is to generate cortical interneuron progenitors in post-mitotic interneuron precursors for mouse embryonic stem cells using a modified embryoid body to monolayer method. This method can help answer key questions in the neuroscience field, such as how sub-types of cortical interneurons achieve their individual fates during development. The main advantage of this technique is that it enables efficient generation of Nkx2.1 expressing interneuron progenitors, as well as methods to enrich for parvalbumin or somatostatin interneuron subgroups.To begin this procedure, plate mitotically inactive MEF feeder cells in MEF media onto tissue culture treated plates. Allow at least 12 hours for them to settle in a cell culture incubator at 37 degrees Celsius before plating the mESCs. If not used immediately, replace the MEF media every three days.Next, add mESCs containing mouse leukemia inhibitory factor to the MEF plates. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide. Passage mESCs using trypsin-EDTA once the dish becomes 70 to 80%confluent, and maintain the mESCs on a MEF feeder layer in mESC media to keep pluripotency.Before starting differentiation, passage the cells at least once onto gelatin-coated plates without MEFs to dilute them out. To do so, prepare gelatin-coated plates by adding 0.1%gelatin and PBS with calcium and magnesium to a tissue culture treated dish and incubate at 37 degrees Celsius for at least 1 hour. Plate 1.5 to two times 10 to the sixth cells on each ten centimeter plate and allow them to expand for two days before beginning differentiation.On DD0, after the mESCs have grown on the gelatin coated plates for two days, aspirate the media and wash the cells once with PBS without calcium and magnesium. Then, add enough trypsin-EDTA to cover the surface of the plates. And place the cells back into the incubator at 37 degrees Celsius for four minutes.After four minutes, quench trypsin-EDTA using two times its volume of mESC media. Transfer the cells to an appropriately sized centrifuge tube, and centrifuge the cells at 200 times g for five minutes. Next, remove the tube and aspirate the media without disturbing the pellet.Resuspend the pellet in one millileter of KSR-N2 media. Subsequently, measure the cell concentration using a hemocytometer, or an automated cell counter. Start growing the cells as embryoid bodies by adding 75, 000 cells per millileter in KSR-N2 media in the non-adherent tissue culture dishes.And incubate them at 37 degrees Celsius. On DD1, prepare for cell landing by coating the tissue culture treated dishes with poly-L-lysine for at least one hour or overnight at 37 degrees Celsius. On DD2, aspirate the poly-L-lysine and coat the plates with laminin overnight at 37 degrees Celsius.On DD3, before beginning EB dissociation, aspirate laminin and allow the plates to completely dry in a tissue culture hood. Afterward, transfer the EB's with the media into a 15 milliliter tube, and centrifuge for three to four minutes at 15 times g or until the EB's have pelleted. Aspirate the media, and add three milliliters of cell detachment solution containing DNase.Then, incubate the sample at 37 degrees Celsius for 15 minutes. Gently flick the tube every three minutes to aid in EB dissociation. Once the EB's are no longer visible, or 15 minutes have elapsed, add six milliliters of KSR-N2 containing DNase, and centrifuge for five minutes at 200 times g.Then, plate the cells in KSR-N2 containing LDN193189, XAV939, and the ROCK inhibitor, Y27632, at 4.5 to five times ten to the fourth cells per centimeter squared. On DD5, change media with KSR-N2 containing FGF2, IGF1, and SSH. Next, prepare tissue culture plates for replating on day 8, using the instructions outlined in the procedures on DD1 and DD2.On DD7, change the media with KSR-N2 containing FGF2, IGF1 and SHH. On DD8, replate the cells by detaching them from the plates with trypsin-EDTA for five minutes at 37 degrees Celsius. Quench trypsin-EDTA using two times its volume with KSR-N2.And centrifuge at 200 times g for five minutes. Filter the cells through a 40 micron filter tube to remove any clumps. Then, replate the cells at 250, 000 cells per centimeter squared in N2-KSR containing FGF2, IGF1 and Y27632 with SHH.On DD10, change the media with KSR-N2 containing SHH. Wash the cells once with PBS without calcium and magnesium, and add pre-warmed, non-trypsin containing cell dissociation reagent, containing DNase to the cells. Place them in the incubator at 37 degrees Celsius for 10 to 30 minutes, or until the cells have detached.Then, gently tap the plates every five minutes to aid the dissociation. On DD5, change the media with KSR-N2 containing FGF2 and IGF1. Next, prepare tissue culture plates for replating on DD8.On DD7, change the media with KSR-N2 containing FGF2 and IGF1. On DD8, replate the cells with smoothened agonist and add atypical PKC inhibitor. On DD10, change the media with KSR-N2 containing aPKCi.On DD11, detach the cells from the plates for FACS. If the cells were not detached for FACS on DD11, change media on DD12 with KSR-N2 containing aPKCi. On DD14, change the media with KSR-N2 containing aPKCi.And on DD16, change the media with KSR-N2 containing aPKCi. Then, on DD17, collect the Nkx-2.1:mCherry positive cells. Shown here are the representative images of immunostaining for Nkx2.1, Nkx2.1:mCherry, and Lhx6:GFP from a differentiation day 12 culture.Note that these images are overlapping channels from the same field of view. This graph shows the quantification of the average percentage of DAPI positive nuclei that express Nkx2.1 at DD12 in culture. And this is a representative FACS plot, demonstrating the four different cell populations that can be isolated using our dual reporter mESC line.Note that mCherry is on the x-axis, and GFP is on the y-axis. Thus, the top right box represents cells that are mCherry-GFP double positive. Here is the timecourse of Nkx2.1:mCherry and Lhx6:GFP induction from DD6 to 16, expressed as the percentage of cells in culture that are either mCherry only expressing, GFP only expressing or mCherry-GFP co-expressing.Once mastered, this protocol can be completed within 17 days from start to finish, if performed properly. While attempting this procedure, it's important to remember to always start with pleuripotent, undifferentiated, healthy appearing stem cells. Following this procedure, other methods, like transplantation into neonatal or adult mouse neocortex can be performed in order answer additional questions regarding interneuron fate determination or the effect of interneuron transplantation on circuit function and animal behavior.After its development, this technique paved the way for researchers in the field of developmental neuroscience to explore factors driving interneuron fate determination in mice, and by extension, patients affected by interneuron related disorders. After watching this video, you should have a good understanding of how to derive Nkx2.1 expressing progenitors, and their post-mitotic progeny from mouse embryonic stem cells. Thanks for watching, and good luck with your experiments.

Research

JoVE Journal

Neuroscience

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