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

Bu protokolü geliştirmeye yardımcı erken çalışmaları için Nkx2.1::mCherry:Lhx6:GFP çift muhabir mESC çizgi gibi Jennifer Tyson, Asif Maroof ve Tim Petros geliştirmek için Qing Xu için minnettarız. Biz de CHOP Akış Sitometresi çekirdek teknik destek için teşekkür ederiz. Bu eser bir NIH R01 MH066912 (SA) ve F30 MH105045-02 (DT) tarafından desteklenmiştir.

Not: Bu protokol için açıklanan çift muhabir mESC hattı (sande@mail.med.upenn.edu) istek üzerine sağlanmaktadır.
1. medyası hazırlama
Not: tüm medya hücre kültüründe kullanmadan önce 37 ° c sıcak.
<ol><li>
Fare embriyonik Fibroblast (MEF) medya (hazırlığı 500 mL)
	<ol>
<li>Dulbecco'nın modifiye kartal orta (DMEM) ve bir 500 mL 0,22 µm gözenek filtre ünitesi filtreden 449 ml 50 mL fetal sığır serum (FBS) ekleyin.</li>
	...

<ol>
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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. 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Bu yöntem J1 kaynaklı mESCs (SCRC-1010) desenlendirme son derece etkili olmakla birlikte, diğer mESC çizgileri ve klonal yalıtır değişken başarı yaşadım. Örneğin, Foxg1::venus mESCs (EB3 kaynaklı; Danjo vd. 13) yanıt kötü bu protokolü ve Foxg1 indüksiyon DD12 tarafından genellikle sırasına % 1-2 olur. Tam olarak anlamıyorum nedeniyle, aynı anda (JQ27 denir) Bu protokol için açıklanan satır olarak izole edildi (JQ59 denir) başka bir Nkx2.1::mCherry:Lhx6::GFP ?...

Fareler ve insanlar içinde kabaca tüm kortikal inhibitör interneurons (CIns) yarısı nerede medial ganglionic hazretleri (MGE) bilinen bir geçici subcortical yapısı içinde kaynaklanan neuroepithelial ataları, CIns ve diğer nöronal ve gliyal alt gruplar transkripsiyon faktörü Nkx2.11,2hızlı. CIn alt gruplar veya alt türlerinden kesişen morfolojik, nörokimyasal, Elektrofizyolojik ve bağlantı özellikleri3,4tarafından tanımlanır. MGE elde edilen CIns gruplandırılabilir çoğunlukla üst üste alt gruplar PV veya SST, hangi ilişkilendirir belirli Elektrofizyolojik ve bağlantı eğilimler5ile ifade onların ifade dayalı. İşlev bozukluğu interneurons, özellikle o PV alt grubu içinde birden çok nöropsikiyatrik bozukluklar ve hastalıklar6,7' karıştığı olmuştur. Bu yöntem genel amacı Mitotik ataları kök hücre kaynaklı ve göçmen öncüleri PV veya SST CIn kader kortikal interneuron biyoloji okuyan ve kullanılmak üzere hücre tabanlı terapiler için zenginleştirilmiş üretmektir.
İnterneuron ataları Nkx2.1 ifade etmek ve onların döl mESCs gelen türetme ölçeklenebilir ve yüksek verimli bir yöntem geliştirdik. Bir Nkx2.1::mCherry:Lhx6::GFP kullanarak çift muhabir mESC kaplamak8, Nkx2.1 ataları ya da onların Lhx6 ifade sonrası Mitotik döl FACS yolu ile izole ve daha sonra aşağı akım uygulamaları bir dizi kullanılacaktır. Sinyal yolları bir dizi, kültür süresi ve neurogenesis modu manipüle ederek, biz fluorescently etiketli interneuron öncüleri aşağı akım uygulamaları bir dizi için uygun milyonlarca alabilirsiniz.
MESCs9,10,11,12,13,14, Wnt üzerinde dayanır bizim yöntem MGE benzeri ataları oluşturmak için birkaç yöntem mevcut olmasına rağmen antagonist XAV-939, Foxg1/Nkx2.1 telencephalic ataları ortak ifade oluşturma özellikle verimli. Buna ek olarak, interneuron ataları veya onların sonrası Mitotik Lhx6 ifade döl çift muhabir sistemimiz üzerinden seçin yeteneği büyük ölçüde farklı ataları ve onların döl üretmek için kapasite artırır.

<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 kortikal interneurons heterojen bir nüfus eksitatör piramidal nöronların çıkışını düzenleyen hem de piramidal nöron topluluklar çıkışlarını eşitleme kritik rol oynarlar hücre vardır. İnterneuron işlevinde açıkları nöropsikiyatrik bozukluklar, şizofreni, otizm ve epilepsi gibi çeşitli karıştığı olmuştur. Embriyonik kök hücrelerden kortikal interneurons türetme sadece kendi geliştirme ve fonksiyon incelenmesi için izin verir, ama kortikal interneuron ile ilgili bozukluklar patogenezinde temel moleküler mekanizmaları görmemizi sağlar. İnterneurons da hayatta, göç ve bütünleşmek içine ana bilgisayar kortikal devresi sonrası nakli, yapım onları kullanmak için ideal aday hücre tabanlı terapiler için olağanüstü kapasitesine sahip. Burada, bir ölçeklenebilir, yüksek verimli, değiştirilmiş embryoid vücut monolayer yöntemi interneuron ataları Nkx2.1 ifade etmek ve onların döl fare embriyonik kök hücreler (mESCs) üzerinden türetme için mevcut. Nkx2.1::mCherry:Lhx6::GFP çift muhabir mESC satırını kullanarak, Nkx2.1 ataları veya onların Lhx6 ifade sonrası Mitotik döl olabilir (FACS) sıralama Floresans aktif hücre ile izole ve daha sonra aşağı akım uygulamaları bir dizi kullanılacaktır. Ayrıca parvalbumin (PV) veya somatostatin için (SST) interneuron alt grupları, hangi-ebilmek var olmak yararlı kader tayini yönlerini çalışmak için veya kullanmak için alt grubu vitaminlerle zenginleştirilmiş interneuron yararlanacak terapötik uygulamalarda zenginleştirmek için yöntemler sağlar nakillerine.

Bu raporda açıklanan iletişim kuralı bizim yayımlanmış protokolleri15,16,17 değiştirilmiş bir versiyonu ve Nkx2.1::mCherry:Lhx6::GFP çift-muhabir mESC hattımız ile kullanım için optimize edilmiştir. DD0-5 DD8 yeniden kaplama ile birlikte, Wnt inhibitörü XAV-939 ekleyerek neyin yukarı tüm DAPI + çekirdeği kültüründe % 50'si de Nkx2.1 (şekil 1A</strong...

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

Bu iletişim kuralı kortikal interneuron ataları ve sonrası Mitotik interneuron öncüleri fare embriyonik kök değiştirilmiş bir embryoid vücut monolayer yöntemi kullanarak hücre oluşturmak için bir yöntem açıklanır. Bu ataları/hareketlenme öncesi ilk belirtiler kullanılan vitro olabilir fluorescently sıralanmış ve kader tayini çalışmak için yenidoğan yeni korteksimiz içine nakledilen veya tedavi uygulamalarında kullanılan.

Fare embriyonik kök hücre farklılaşma içine kortikal Interneuron Kara filmin tarih öncesi

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

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

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

Fare önbeyin içine Ultrason Rehberliğinde Mikroenjeksiyon In Utero E9.5 az

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

Nörobilim

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

Yetişkin Hipokampal Arctic Zemin Sincap Nöral Kök Hücre Büyüme ve Farklılaşma

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

Embriyonik Fare Hipokampal ve Kortikal nöronlar primer Nucleofection ve Kültür

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

Neurosphere Testi kullanarak Embriyonik Fare Nöral Kök Hücre Kültürü oluşturulması

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

Atılan İnsan Fetal Kortikal Doku Sinir Kök Hücre Üretimi

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

Spinal Kordonlar nörotropik Mouse Hepatit Virüs ile Etkilenmiş Fare Fare Nöral Kök Hücre Transplantasyon Cerrahi

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

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

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

İnsan Pluripotent Kök Hücreleri dopaminerjik Nöron Farklılaşma Yönetmen

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

Erken Postnatal fare beyin içine Interneuron öncüleri Homochronic nakli

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