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

Siamo grati a Qing Xu per sviluppare il Nkx2.1::mCherry:Lhx6:GFP dual reporter mESC linea così come Jennifer Tyson, Asif Maroof e Tim Petros per i loro primi lavori su aiutando a sviluppare questo protocollo. Ringraziamo anche il nucleo di cytometry di flusso CHOP per assistenza tecnica. Questo lavoro è stato supportato da un NIH R01 MH066912 (SA) e F30 MH105045-02 (DT).

Nota: La linea di mESC dual reporter descritta in questo protocollo è disponibile su richiesta (sande@mail.med.upenn.edu).
1. Media preparazione
Nota: Caldo tutti i media a 37 ° C prima dell'uso nella coltura delle cellule.
<ol><li>
Media dei fibroblasti embrionali del mouse (MEF) (per la preparazione di 500 mL)
	<ol>
<li>Aggiungere 50 mL di siero fetale bovino (FBS) 449 mL di Medium (DMEM dell'Aquila per volta) di Dulbecco e filtro attraverso u...

<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>

Mentre questo metodo è altamente efficace a patterning mESCs J1-derivato (SCRC-1010), abbiamo sperimentato successo variabile con altre linee di mescita e isolati clonali. Per esempio, Foxg1::venus mESCs (EB3-derivato; Dergano et al. 13) rispondono male a questo protocollo e Foxg1 induzione da DD12 è tipicamente dell'ordine di 1-2%. Per motivi che non comprendiamo pienamente, clone di Nkx2.1::mCherry:Lhx6::GFP un altro reporter dual (chiamato JQ59) che è stato isolato simultaneamente c...

In entrambi i topi e gli esseri umani, circa la metà di tutto corticale interneuroni inibitori (CIns) hanno origine all'interno di una struttura subcortical transitoria, conosciuta come l'eminenza gangliare mediale (MGE), dove i progenitori di neuroepithelial CIns e altri un neurone e glial sottogruppi esprimono il fattore di trascrizione NKX 2.11,2. Sottogruppi di CIn o sottotipi sono definiti da intersecanti morfologiche, neurochimici, elettrofisiologici e connettività caratteristiche3,4. Il CIns MGE-derivati possono essere raggruppati in sottogruppi per lo più non sovrapposte basati sulla loro espressione di PV o SST, l'espressione di cui correla con particolare elettrofisiologici e connettività di tendenze5. Disfunzione di interneuroni, specialmente quelli nel sottogruppo PV, è stata implicata in più neuropsichiatrici disturbi e malattie6,7. L'obiettivo generale di questo metodo è quello di produrre cellule staminali derivate mitotici progenitori e precursori migratori arricchiti per destino o PV o CIn SST per studiare biologia Interneurone corticale e per uso in terapie basate sulle cellule.
Abbiamo sviluppato un metodo scalabile, altamente efficiente per la derivazione di NKX 2.1-esprimendo Interneurone progenitori e loro progenie da mESCs. Utilizzando un Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC linea8, NKX 2.1 progenitori o loro progenie post-mitotici esprimenti Lhx6 può essere isolato tramite FACS e successivamente utilizzato in una serie di applicazioni a valle. Manipolando un numero di vie di segnalazione, durata della cultura e la modalità della neurogenesi, possiamo ottenere milioni di precursori fluorescente contrassegnati Interneurone adatti per una miriade di applicazioni a valle.
Anche se esistono diversi altri metodi per la generazione di MGE-come progenitori da mESCs9,10,11,12,13,14, il nostro metodo, che si basa su Wnt antagonista XAV-939, è particolarmente efficiente nella generazione Foxg1/NKX 2.1 cheesprimono telencefalici progenitori. Inoltre, la possibilità di selezionare per progenitori Interneurone o loro progenie che esprimono Lhx6 post-mitotici tramite il nostro sistema di dual reporter, migliora notevolmente la capacità di generare distinte dei progenitori e la loro progenie.

<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

Corticali GABAergici sono una popolazione eterogenea di cellule che giocano un ruolo critico in che regolano l'uscita dei neuroni piramidali eccitatori, nonché la sincronizzazione le uscite dell'ensemble neurone piramidale. I deficit nella funzione Interneurone sono stati implicati in una varietà di disordini neuropsichiatrici, compreso la schizofrenia, autismo ed epilessia. La derivazione di interneuroni corticali da cellule staminali embrionali non solo consente lo studio del loro sviluppo e la funzione, ma fornisce la comprensione nei meccanismi molecolari alla base della patogenesi dei disordini corticali da Interneurone. Interneuroni hanno anche la notevole capacità di sopravvivere, migrare e integrare in host circuiti corticali post-trapianto, che li rende i candidati ideali per l'utilizzo nelle terapie basate sulle cellule. Qui, presentiamo un metodo corpo-a-monostrato embryoid scalabile, altamente efficiente, modificato per la derivazione di NKX 2.1-esprimendo Interneurone progenitori e loro progenie da cellule staminali embrionali di topo (mESCs). Utilizzando una linea di mESC reporter dual Nkx2.1::mCherry:Lhx6::GFP, NKX 2.1 progenitori o loro progenie post-mitotici esprimenti Lhx6 può essere isolato tramite fluorescenza-attivato delle cellule ordinano (FACS) e successivamente utilizzato in una serie di applicazioni a valle. Forniamo anche metodi per arricchire per parvalbumina (PV) o somatostatina sottogruppi di Interneurone (SST), che può essere utile per lo studio di aspetti della determinazione di destino o per l'uso in applicazioni terapeutiche che beneficerebbero Interneurone arricchita di sottogruppo trapianti.

Il protocollo descritto in questo documento è una versione modificata di nostri protocolli pubblicati15,16,17 ed è stato ottimizzato per l'uso con la nostra linea di dual-reporter mESC Nkx2.1::mCherry:Lhx6::GFP. Aggiungendo l'inibitore di Wnt XAV-939 da DD0-5, combinato con ri-placcatura a DD8, raggiungiamo robusto NKX 2.1 induzione, in cui verso l'alto di 50% di tutti i nuclei DAPI + nella cul...

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

Questo protocollo descrive un metodo per la generazione di corticale Interneurone progenitori e precursori post-mitotici Interneurone da cellule staminali embrionali di topo usando un metodo modificato embryoid corpo-a-monostrato. Questi progenitori/precursori possono essere usati in vitro o fluorescente ordinati e trapiantate nel neocortex neonatale per lo studio della determinazione di destino o usati in applicazioni terapeutiche.

Differenziazione delle cellule staminali embrionali di topo in precursori Interneurone corticale

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

Ultrasound-Guided Microiniezione nel proencefalo mouse In Utero A E9.5

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

Ricerca

Neuroscienze

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

Crescita e la differenziazione degli adulti ippocampale suolo artico Cellule Staminali neurali Squirrel

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 e cultura primaria di neuroni embrionali di topo ippocampale e corticale

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

Stabilire embrionali di topo Neural Stem Cell Culture del saggio di neurosfere

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

Generazione di cellule staminali neurali da scartate tessuto fetale umano corticale

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

Il trapianto chirurgico di cellule staminali neurali del mouse nel midollo spinale di topi infettati con virus dell&#39;epatite neurotropi mouse

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

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

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

Isolamento e la coltura di astrociti corticali mouse

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

Regia neuroni dopaminergici differenziazione da cellule staminali pluripotenti umane

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 trapianto di precursori Interneurone in cervelli di topo postnatale precoce

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