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>
		<li>1 mL antimikrobiyal ajan (50 mg/mL) filtrasyon sonra ekleyin. 1 ay veya aliquot ve ≤ dükkanında 4 ° C'de medya saklamak-20 ° c</li>
	</ol>
</li>
	<li>
N2 medya (hazırlığı 500 mL)
	<ol>
<li>5 mL L-alanin-L-glutamin ekleyin (100 x) ve 500 µL 2-Mercaptoethanol (55 mM) 489.5 ml DMEM: Besin karışımı F-12 (DMEM/F-12) ve 500 mL 0,22 µm gözenek filtre ünitesi filtreden.</li>
		<li>1 mL antimikrobiyal ajan (50 mg/mL) ve 5 mL N2 ek-B filtrasyon sonra ekleyin. Medya 4 ° c ilâ 1 ay boyunca karanlıkta saklayın.</li>
	</ol>
</li>
	<li>
(500 mL hazırlığı) serum-Alerjik büyüme orta (KSR)
	<ol>
<li>75 mL serum-Alerjik orta ek, 5 mL L-glutamin ekleyin (100 x), 5 mL MEM sigara-esansiyel Amino asitler (MEM-NEAA) (100 x), ve 500 µL 2-Mercaptoethanol (55 mM) 413.5 mL sigara-glutamin içeren DMEM ve filtre 500 mL 0,22 µm gözenek filtre birimi üzerinden.</li>
		<li>1 mL antimikrobiyal ajan (50 mg/mL) filtrasyon sonra ekleyin. Medya 4 ° c ilâ 1 ay için saklayın.</li>
	</ol>
</li>
	<li>
Fare embriyonik kök hücre medya (hazırlığı 500 mL)
	<ol>
<li>75 mL kök hücre sınıf FBS, 5 mL MEM-NEAA ekleyin (100 x), 5 mL L-glutamin (100 x), ve 500 µL 2-Mercaptoethanol (55 mM) 413.5 mL sigara-glutamin içeren DMEM ve filtre 500 mL 0,22 µm gözenek filtre birimi üzerinden.</li>
		<li>1 mL antimikrobiyal ajan (50 mg/mL) filtrasyon sonra ekleyin. Medya 4 ° C'de 1 ay veya aliquot ve ≤ mağazasında için karanlıkta saklamak-20 ° c</li>
	</ol>
</li>
</ol>2. Kültür mESCs
<ol><li>Plaka mitotically etkin olmayan MEF besleyici hücreler MEF medya doku kültürü üzerine 3-4 x 104 hücreleri/cm2tabak tedavi. En az 12 h onları mESCs kaplama daha önce bir hücre kültür kuluçka ≥ % 95 bağıl nem ve % 5 CO2 37 ° C'de yerleşmek izin verir. Hemen değil kullandıysanız, her 3 günde MEF ortamı değiştirin. MEFs kaplama 7 gün içinde kullanılmayan eğer atın.</li>
	<li>MESCs (1-4 x 104 hücreleri/cm2yoğunluk aralığı) MEF plakaları fare lösemi inhibitör faktör (mLIF) (1000 U/mL) içeren mESC medya ekleyin. ≥ % 95 bağıl nem ve % 5 CO237 ° C'de hücreler kuluçkaya.</li>
	<li>Geçiş tripsin-EDTA (% 0.05 tripsin) kullanarak mESCs (1:5-1:10) çanak olur sonra ~ % 70-80 birleşmesi veya koloniler dokunmaya başlıyor. Bu genellikle 2 günde ortaya çıkar, ancak ilk kaplama yoğunluğu bağlı olarak 4 veya 5 gün sürebilir.</li>
	<li>MESCs mESC orta pluripotency tutmak için MEF besleyici katmandaki korumak.</li>
	<li>
Farklılaşma başlamadan önce en az bir kez üzerine jelatin kaplı plakaları olmadan MEFs sulandırmak için MEFs hücreleri geçiş.
	<ol>
<li>% 0.1 ekleyerek jelatin kaplı plakalar fosfat tamponlu tuz (PBS) ile Ca2 +/Mg2 + bir doku kültürü tedavi çanak içinde jelatin hazırlamak ve en az 1 h. plaka 1.5-2 x 106 hücreler/10 cm tabak için 37 ° C'de bırakarak (2.7-3.6 x 104 hücre/cm2) ve hücre farklılaşması başlamadan önce 2 gün boyunca genişletmek izin verir.</li>
	</ol>
</li>
</ol>3. mESCs MGE benzeri Telencephalic ataları doğru ayırt
<ol><li>
Farklılaşma gün (DD) 0 "hücreleri float" =
	<ol>
<li>MESCs büyüdü sonra jelatin kaplı levha 2 gündür medya Aspire edin ve PBS olmadan Ca2 +/Mg2 +bir kez hücrelerle yıkayın. Yeterli tripsin-plaka (tipik olarak 4 mL tripsin-EDTA bir 10 cm doku kültürü yemek için) yüzeyine kapak ve geri içine ≥95% bağıl nem ve % 5 CO2 37 ° C'de kuluçka makinesi 4 dk için hücreleri yerleştirmek için EDTA (% 0.05 tripsin) ekleyin.</li>
		<li>4 dk sonra mESC medya ile 2 kez birimi kullanan tripsin-EDTA gidermek. Hücreleri bir uygun ölçekli santrifüj tüpü aktarmak ve hücreleri 200 x g 5 min için de santrifüj kapasitesi. 5 dk sonra tüpü çıkarması ve medya Pelet bozmadan Aspire edin. BMP inhibitörü LDN-193189 içeren Pelet 1 ml KSR:N2 medya (1:1) resuspend (250 nM) ve Wnt inhibitörü XAV-939 (10 µM).</li>
		<li>Bir hemasitometre veya otomatik hücre sayacı kullanarak hücre konsantrasyonu ölçmek. LDN-193189 içeren KSR:N2 medya (1:1) 75.000 hücre/mL ekleyerek olarak embryoid organları (EBs) hücreleri büyüyen Başlat (250 nM) ve XAV-939 (10 µM) yapışık olmayan doku kültürü yemekleri. ≥ % 95 bağıl nem ve % 5 CO237 ° C'de hücreler kuluçkaya.</li>
	</ol>
</li>
	<li>DD1 üzerinde "kaplama doku kültürü tedavi yemekleri ile Poli-L-lizin (10 µg/mL PBS ile Ca2 +/Mg2 +) tarafından bir gecede açılış" hücre (O/N) ≥ % 95 bağıl nem ile 37 ° C'de veya en az 1 h için hazırlayın.</li>
	<li>DD2, Poli-L-lizin Aspire edin ve laminin (10 µg/mL PBS ile Ca2 +/Mg2 +) ile plakalar ceket O/N ≥ % 95 bağıl nem ile 37 ° C'de. Not: O/N optimal, gibi küçük ise 2 h yeterli olabilir. Tabak diğer gün kullanılmazsa, laminin Aspire edin, PBS ile Ca2 +/Mg2 +değiştirin ve 4 ° C'de 2 hafta için saklamak.</li>
	<li>
DD3 ("Kara hücreleri")
	<ol>
<li>EB ayrılma işlemine başlamadan önce laminin Aspire edin ve tabakaları bana izin tamamen kuru bir doku kültürü Hood. Parlak veya gözle görülür ıslak görünen plakaları kullanmayın. EBs medya ile 15 mL tüp ve 15 g x veya EBs pelleted kadar 3-4 dk santrifüj içine aktarın.</li>
	</ol>
</li>
	<li>Medya Aspire edin ve 3 mL Dnaz içeren hücre dekolmanı çözümü ekleyin (2 U/mL) ve ≥ % 95 bağıl nem ile 37 ° C'de kuluçkaya ve % 5 CO2 15 dakika süreyle yavaşça EB ayrılma yardım için tüp her 3 dakikada fiske.</li>
	<li>EBs artık görünür veya 15dk geçen kez Dnaz içeren 6 mL KSR:N2 (1:1) eklemek (1 U/mL) ve 200 x g de 5 dk santrifüj.</li>
	<li>KSR:N2 (1:1) LDN-193189 içeren hücrelerde plaka (250 nM), XAV-939 (10 µM) ve ROCK inhibitörü Y-27632 (10 µM) 4.5-5 x 104 hücreleri/cm2.</li>
</ol>4. SST zenginleştirici Protokolü: "DD12 GFP yüksek Sonic Kirpi (SHH)" (Adım 3.7 devamı)
<ol><li>DD5 üzerinde FGF-2 içeren medya KSR:N2 (1:1) ile değiştirmek (10 ng/mL), IGF-1 (20 ng/mL) ve SSH (50 ng/mL).</li>
	<li>Gün 8 (Adım 4.4) yeniden kaplama için doku kültürü tabak hazırlamak 3.2-3.3 adımlarda açıklanan yönergeleri kullanarak.</li>
	<li>DD7 üzerinde FGF-2 içeren medya KSR:N2 (1:1) ile değiştirmek (10 ng/mL), IGF-1 (20 ng/mL) ve ŞŞŞ (50 ng/mL).</li>
	<li>
DD8 üzerinde hücreleri yeniden plaka. Not: Bu adım gerekli değildir, ancak hücreleri yeniden kaplama erken farklılaşmış, istenmeyen hücre tipleri azaltabilir. Yeniden kaplama aynı zamanda aşağı akım immunohistokimyasal analizleri yapmak zor hücre topları keser ve sonraki zaman noktalarda FACS verimliliğini artırır.<ol>
<li>Hücreleri yeniden plaka, plaka ile tripsin-EDTA (% 0.05 tripsin) hücrelerden ≥95% bağıl nem ve % 5 CO237 ° C'de 5 min için ayırmak için. KSR:N2 ile 2 kez birimi kullanan tripsin-EDTA gidermek ve 5 dakika süreyle 200 x g, santrifüj filtre hücreleri herhangi bir kümeleri kaldırmak için 40 µm filtre tüpünden. FGF-2 içeren hücreler 250.000 hücreler/cm2 N2/KSR (1:1)'de yeniden plaka (10 ng/mL), IGF-1 (20 ng/mL) ve Y-27632 (10 µM) ŞŞŞ ile (50 ng/mL). Not: Bunun yerine, karar DD8 sayfasındaki hücreleri yeniden plaka değil yaptıysanız, medya ŞŞŞ içeren KSR:N2 ile değiştirmek (50 ng/mL) DD12 DD7 her 2 gün ve IGF-1 eklemeye devam edin (20 ng/mL) ve FGF-2 (10 ng/mL) DD9 kadar.</li>
	</ol>
</li>
	<li>DD10 üzerinde medya ŞŞŞ içeren KSR:N2 ile değiştirmek (50 ng/mL).</li>
	<li>
DD12 üzerinde SST alt için zenginleştirilmiş hücreleri elde etmek için FACS Lhx6::GFP belirlemek için kullanın-sadece hücreleri ifade.
	<ol>
<li>Hücreleri ayırmak için bir sigara tripsin içeren hücre-ayrılma reaktif tripsin-EDTA yerine kullanın. Hücreleri bir kez PBS olmadan Ca2 +/Mg2 +ile yıkayın. Dnaz içeren önceden ısıtılmış tripsin sigara içeren hücre-ayrılma reaktif ekleyin (2 U/mL) hücreleri. Kuluçka makinesi 10-30 dk ya kadar hücreleri bağlantısız ≥95% bağıl nem ve % 5 CO2 37 ° C'de yer onları. Yavaşça plakaları her 5 dk ayrılma yardım için dokunun. Not: DD11-12 kültürler için sadece 10-15 dk gerekli olabilir. DD15-16 kültürler için 30 dakika veya daha fazla tam ayrılma için gerekli olabilir.</li>
		<li>Hücreleri tamamen tabağını kaldırdı, standart protokolleri kullanarak FACS izolasyon için ilerlemek veya diğer aşağı akım analizleri hücrelerdeki kullanmak.</li>
	</ol>
</li>
</ol>5. PV zenginleştirici Protokolü: "DD11/DD17 mCherry + aPKCi"--dan adım 3.7 (devamı)
<ol><li>DD5 üzerinde FGF-2 içeren medya KSR:N2 (1:1) ile değiştirmek (10 ng/mL) ve IGF-1 (20 ng/mL).</li>
	<li>DD8 yeniden kaplama için doku kültürü tabak hazırlamak (Adım 4.4) kullanma yönergeleri özetlenen adımlar 3.2-3.3.</li>
	<li>DD7 üzerinde FGF-2 içeren medya KSR:N2 (1:1) ile değiştirmek (10 ng/mL) ve IGF-1 (20 ng/mL).</li>
	<li>DD8 üzerinde hücreleri aşağıdaki istisnalar 4.4. adımda açıklandığı gibi yeniden plaka: hücreleri yeniden kaplama, ŞŞŞ ile smoothened agonist yerine (SAG; 30 nM) ve atipik PKC inhibitörü (aPKCi; 2 µM) ekleyin. Birlikte, yeniden hassasiyette medya ele alındığında şimdi N2/KSR (1:1) FGF-2 içeren (10 ng/mL), IGF-1 (20 ng/mL), Y-27632 (10 µM), SAG (30 nM) ve aPKCi (2 µM).</li>
	<li>DD10 üzerinde medya aPKCi (2 µM) içeren KSR:N2 ile değiştirin.</li>
	<li>
DD11 Not: Bu noktada, Nkx2.1::mCherry+ hücreleri PV CIns olmak için zenginleştirilmiş.<ol>
<li>4.6. adımda belirtilen aynı iletişim kuralını kullanarak FACS için plaka hücrelerden ayırmak. Karar daha sonra bir farklılaşma gün Nkx2.1::mCherry+ hücreleri toplamak için bu adımı dikkate almayın.</li>
	</ol>
</li>
	<li>DD12 üzerinde medya aPKCi (2 µM) içeren KSR:N2 ile değiştirin. DD14 üzerinde medya aPKCi (2 µM) içeren KSR:N2 ile değiştirin. DD16 üzerinde medya aPKCi (2 µM) içeren KSR:N2 ile değiştirin. DD17 üzerinde 4.6. adımda belirtilen aynı iletişim kuralını kullanarak Nkx2.1::mCherry+ hücreleri toplamak.</li>
</ol>6. PV zenginleştirici Protokolü: "DD17 mCherry düşük ŞŞŞ" (Adım 3.7 devamı)
<ol><li>DD5 üzerinde FGF-2 içeren medya KSR:N2 (1:1) ile değiştirmek (10 ng/mL) ve IGF-1 (20 ng/mL). DD7 üzerinde FGF-2 içeren medya KSR:N2 (1:1) ile değiştirmek (10 ng/mL) ve IGF-1 (20 ng/mL).</li>
	<li>
DD9 üzerinde medya KSR:N2 (1:1) ile değiştirin. Bu noktadan itibaren hiçbir ek büyüme faktörleri gereklidir.
	<ol>
<li>2 günde DD17 hücrelerdeyse hasat kadar medya değiştirmeye devam.</li>
	</ol>
</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+interneuron+transplants+to+study+development+and+treat+disease.">GABAergic interneuron transplants to study development and treat disease.</a> Trends Neurosci. 37, 169-177 (2014).</li><li>Donegan, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+interneuron+transplants+as+a+treatment+for+schizophrenia:+preclinical+validation+in+a+rodent+model.">Stem cell-derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model.</a> Mol Psychiatry. , (2016).</li><li>Deglincerti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+human+embryonic+stem+cells+on+micropatterns.">Self-organization of human embryonic stem cells on micropatterns.</a> Nat Protoc. 11, 2223-2232 (2016).</li><li>Chambers, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nat Biotechnol. 27, 275-280 (2009).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+naive-like+human+pluripotent+stem+cells+based+on+HERVH+expression.">Isolation and cultivation of naive-like human pluripotent stem cells based on HERVH expression.</a> Nat Protoc. 11, 327-346 (2016).</li><li>Zeltner, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+the+biology+of+disease+severity+in+a+PSC-based+model+of+familial+dysautonomia.">Capturing the biology of disease severity in a PSC-based model of familial dysautonomia.</a> Nat Med. 22, 1421-1427 (2016).</li><li>Blahos, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+site+on+the+Galpha+-protein+that+recognizes+heptahelical+receptors.">A novel site on the Galpha -protein that recognizes heptahelical receptors.</a> J Biol Chem. 276, 3262-3269 (2001).</li></ol>

Yazarlar ifşa gerek yok.

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 ...

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Neuroscience

10.3K Görüntüleme.  University of Pennsylvania. 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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

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