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

Wir sind dankbar, Qing Xu für die Entwicklung der Nkx2.1::mCherry:Lhx6:GFP dual Reporter mESC Linie und Jennifer Tyson, Asif Maroof, sowie Tim Petros für ihre frühen Arbeiten zu helfen, dieses Protokoll zu entwickeln. Wir danken auch den CHOP Flow Cytometry Kern für technische Hilfe. Diese Arbeit wurde von einem NIH R01 MH066912 (SA) und F30 MH105045-02 (DT) unterstützt.

Hinweis: Die dual Reporter mESC Linie beschrieben in diesem Protokoll sind auf Anfrage möglich (sande@mail.med.upenn.edu).
(1) Media-Vorbereitung
Hinweis: Warm alle Medien auf 37 ° C vor der Verwendung in der Zellkultur.
<ol><li>
Maus embryonalen Fibroblasten (MEF) Medien (für die Zubereitung 500 mL)
	<ol>
<li>449 mL Dulbeccos geändert Eagle Medium (DMEM) und Filter durch eine Filteranlage 500 mL 0,22 µm Pore 50 mL fetalen bovine Serum (FBS) ...

<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. 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=Petilla+terminology:+nomenclature+of+features+of+GABAergic+interneurons+of+the+cerebral+cortex.">Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.</a> Nature reviews. Neuroscience. 9, 557-568 (2008).</li><li>DeFelipe, 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=New+insights+into+the+classification+and+nomenclature+of+cortical+GABAergic+interneurons.">New insights into the classification and nomenclature of cortical GABAergic interneurons.</a> Nature reviews. Neuroscience. 14, 202-216 (2013).</li><li>Xu, X., Roby, K. D., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+characterization+of+inhibitory+mouse+cortical+neurons:+three+chemically+distinct+classes+of+inhibitory+cells.">Immunochemical characterization of inhibitory mouse cortical neurons: three chemically distinct classes of inhibitory cells.</a> J Comp Neurol. 518, 389-404 (2010).</li><li>Inan, M., Petros, T. 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=Losing+your+inhibition:+Linking+cortical+GABAergic+interneurons+to+schizophrenia.">Losing your inhibition: Linking cortical GABAergic interneurons to schizophrenia.</a> Neurobiol Dis. 53, 36-48 (2013).</li><li>Benes, F. M., Berretta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+interneurons:+implications+for+understanding+schizophrenia+and+bipolar+disorder.">GABAergic interneurons: implications for understanding schizophrenia and bipolar disorder.</a> Neuropsychopharmacology. 25, 1-27 (2001).</li><li>Tyson, J. A., Goldberg, E. M., Maroof, A. M., Petros, T. 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=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>Au, E., 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+modular+gain-of-function+approach+to+generate+cortical+interneuron+subtypes+from+ES+cells.">A modular gain-of-function approach to generate cortical interneuron subtypes from ES cells.</a> Neuron. 80, 1145-1158 (2013).</li><li>Petros, T. J., Maurer, C. W., 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=Enhanced+derivation+of+mouse+ESC-derived+cortical+interneurons+by+expression+of+Nkx2.1.">Enhanced derivation of mouse ESC-derived cortical interneurons by expression of Nkx2.1.</a> Stem Cell Res. 11, 647-656 (2013).</li><li>Cambray, S., 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=Activin+induces+cortical+interneuron+identity+and+differentiation+in+embryonic+stem+cell-derived+telencephalic+neural+precursors.">Activin induces cortical interneuron identity and differentiation in embryonic stem cell-derived telencephalic neural precursors.</a> Nat Commun. 3, 841 (2012).</li><li>Chen, Y. 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=Use+of+"MGE+Enhancers"+for+Labeling+and+Selection+of+Embryonic+Stem+Cell-Derived+Medial+Ganglionic+Eminence+(MGE)+Progenitors+and+Neurons.">Use of "MGE Enhancers" for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons.</a> PloS one. 8, e61956 (2013).</li><li>Danjo, T., 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=Subregional+specification+of+embryonic+stem+cell-derived+ventral+telencephalic+tissues+by+timed+and+combinatory+treatment+with+extrinsic+signals.">Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 31, 1919-1933 (2011).</li><li>Watanabe, K., 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+of+telencephalic+precursors+from+embryonic+stem+cells.">Directed differentiation of telencephalic precursors from embryonic stem cells.</a> Nat Neurosci. 8, 288-296 (2005).</li><li>Tyson, J. 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. 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>

Während diese Methode ist sehr effektiv bei der Musterung J1 abgeleitet mESCs (SCRC-1010), erlebten wir Variablen Erfolg mit anderen mESC Linien und klonale Isolate. Zum Beispiel, Foxg1::venus mESCs (EB3 abgeleitet; Danjo Et al. 13) reagieren schlecht auf dieses Protokoll und Foxg1 Induktion durch DD12 ist in der Regel in der Größenordnung von 1 %-2 %. Aus Gründen, die wir nicht vollständig verstehen, produziert ein weiteres Nkx2.1::mCherry:Lhx6::GFP dual Reporter Klon (genannt JQ59)...

Bei Mäusen und Menschen, rund die Hälfte aller kortikalen hemmenden Interneuronen (CIns) stammen im Rahmen einer vorübergehenden subkortikale Struktur bekannt als mediale ganglionic Eminenz (MGE), wo neuroepithelialer Vorfahren der CIns und anderen neuronalen und Gliazellen Untergruppen Ausdrücken der Transkriptionsfaktor Nkx2.11,2. CIn Untergruppen oder Subtypen sind durch sich überschneidende morphologische, neurochemische, elektrophysiologische und Konnektivität Merkmale3,4definiert. MGE-abgeleitete CIns in gruppiert werden können meist nicht überlappende Untergruppen basierend auf ihren Ausdruck der PV oder SST, der Ausdruck der welche korreliert mit bestimmten elektrophysiologische und Konnektivität Tendenzen5. Dysfunktion von Interneuronen, insbesondere in der PV-Untergruppe hat in mehreren-neuropsychiatrischen Störungen und Krankheiten6,7verwickelt. Das übergeordnete Ziel dieser Methode ist, mitotischen Vorläuferzellen Stammzellen abgeleitet und wandernden Vorstufen für PV oder SST CIn Schicksal für kortikale Interneuron Biologie zu studieren und für den Einsatz in zellbasierten Therapien angereichert zu produzieren.
Wir haben eine skalierbare und hocheffiziente Methode zur Ableitung von Nkx2.1 exprimierenden Interneuron Vorfahren und deren Nachkommen von mESCs entwickelt. Mit einem Nkx2.1::mCherry:Lhx6::GFP dual Reporter mESC Zeile8, Nkx2.1 Vorfahren oder Lhx6 exprimierenden Post-mitotischen Nachkommen über FACS isoliert und anschließend in einer Reihe von downstream-Anwendungen verwendet werden können. Durch die Manipulation einer Anzahl der Signalwege, Dauer der Kultur und Modus der Neurogenese, erhalten wir Millionen von Eindringmittel beschrifteten Interneuron Vorstufen für eine Vielzahl von downstream-Anwendungen geeignet.
Obwohl einige andere Methoden gibt es zur Erzeugung von MGE-ähnlichen Vorfahren aus mESCs9,10,11,12,13,14, unsere Methode, die stützt sich auf die Wnt Antagonist XAV-939 ist besonders effizient zu generieren Foxg1/Nkx2.1 Co Teleenzephalischer Vorfahren zum Ausdruck zu bringen. Stark erhöht die Fähigkeit, für Interneuron Stammväter oder Post-mitotischen Lhx6 exprimierenden Nachkommen über unser dual Reporter-System wählen die Fähigkeit, verschiedene Vorfahren und deren Nachkommen zu erzeugen.

<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

Kortikale GABAergen Interneuronen sind eine heterogene Bevölkerung der Zellen, die kritische Rollen bei der Regelung des Ausgang des exzitatorischen pyramidale Neuronen sowie synchronisieren die Ausgänge der pyramidalen Neuron Ensembles spielen. Defizite im Interneuron Funktion haben in einer Vielzahl von neuropsychiatrischen Störungen wie Schizophrenie, Autismus und Epilepsie verwickelt. Die Ableitung der kortikale Interneurone aus embryonalen Stammzellen nicht nur für das Studium ihrer Entwicklung und Funktion ermöglicht, sondern bietet einen Einblick in die molekularen Mechanismen der Pathogenese der kortikalen Interneuron-Erkrankungen. Interneuronen haben auch die bemerkenswerte Fähigkeit, überleben, Migration und Integration in Host kortikalen Schaltung nach Transplantation, so dass sie ideale Kandidaten für den Einsatz in zellbasierten Therapien. Hier präsentieren wir Ihnen eine skalierbare, hocheffiziente, modifizierte Embryoid Körper-Monolayer-Methode zur Ableitung von Nkx2.1 exprimierenden Interneuron Vorfahren und deren Nachkommen aus embryonalen Stammzellen der Maus (mESCs). Mit einem Nkx2.1::mCherry:Lhx6::GFP dual Reporter mESC Linie, Nkx2.1 Vorfahren oder Lhx6-mit dem Ausdruck nach dem mitotischen Nachkommen isoliert über Fluoreszenz-aktivierte Zelle sortieren (FACS) und anschließend in einer Reihe von downstream-Anwendungen verwendet. Wir bieten auch Methoden bereichern Parvalbumin (PV) oder Somatostatin (SST) Interneuron Untergruppen, die für das Studium Aspekte des Schicksal Bestimmung oder für den Einsatz in therapeutischen Anwendungen möglicherweise hilfreich, die von Interneuron Untergruppe angereicherte profitieren würde Transplantationen.

Das Protokoll in diesem Dokument beschriebenen ist eine modifizierte Version von unseren veröffentlichten Protokolle15,16,17 und wurde für den Einsatz mit unseren Nkx2.1::mCherry:Lhx6::GFP Dual-Reporter mESC optimiert. Durch das Hinzufügen der Wnt-Inhibitors XAV-939 DD0-5, kombiniert mit neu bei DD8 Beschichtung, erreichen wir robuste Nkx2.1 Induktion, wobei mehr als 50 % der alle DAPI + Kerne...

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

Dieses Protokoll beschreibt eine Methode zur Erzeugung von kortikalen Interneuron Stammväter und Post-mitotische Interneuron Vorstufen aus embryonalen Stammzellen der Maus mit einer veränderten Embryoid Körper-Monolayer-Methode. Diese Vorfahren/Vorstufen können werden verwendet in-vitro- Eindringmittel sortiert und in Neugeborenen Neokortex für das Studium Schicksal Bestimmung verpflanzt oder in therapeutischen Anwendungen eingesetzt.

Differenzierung der embryonalen Stammzellen der Maus in kortikalen Interneuron Vorstufen

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

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

Ultraschall-gesteuerte Mikroinjektion in der Maus Vorderhirnneuronen In Utero In E9.5

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

Forschung

Neurowissenschaften

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

Wachstum und Differenzierung von adulten Hippocampus 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 ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Nucleofection und Primärkultur von embryonalen Maus-Hippocampus und kortikalen Neuronen

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

Gründung Embryonale Maus Neural Stem Cell Culture Mit dem Neurosphäre 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 ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Generierung von neuralen Stammzellen aus menschlichen Föten Ausrangierte Rindengewebe

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

Chirurgische Transplantation von Maus Neural Stem Cells in den Rückenmark von Mäusen mit neurotropen Maus Hepatitis Virus infiziert

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

Isolierung und Kultivierung von Zellen der Neuralleiste aus embryonalen murinen Neuralrohr

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

Isolation and Culture of Mouse Cortical Astrocytes

Isolation und Kultur von Maus Kortikale Astrozyten

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Regie dopaminergen Neuron Differenzierung von humanen pluripotenten Stammzellen

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

Homochronic Transplantation von Interneuron Vorstufen in frühen postnatalen Mäusegehirnen

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