Diese Arbeit wurde von der National Natural Science Foundation of China (Nr. 31501099) und der Middle-aged and Young of the Education Department of Hubei Province, China (Nr. Q20191104). Und wir danken Professor Wensheng Deng von der Wuhan University of Science and Technology für die Bereitstellung der mausembryonalen Stammzelllinien A2lox.

1. Maus embryonale Stammzellkultur
<ol><li>Bereiten Sie 0,1% gelatinebeschichtete Zellkultur-Gerichte oder Teller zu.<ol>
<li>2 ml sterilisierte 0,1% Gelatine (0,1% w/v in Wasser) zu 60 mm Zellkulturschalen hinzufügen. Schaukeln Sie sanft, um eine gleichmäßige Beschichtung der Zellkulturgerichte zu gewährleisten.</li>
		<li>Die Gerichte bei 37 °C in einen 5% CO2-Inkubator geben und 1 h beschichten lassen.</li>
		<li>Entfernen Sie die 0,1% Gelatinelösung, bevor Sie die Zellen säen. HINWEIS: Nach dem Entfernen der Gelatine ist es nicht erforderlich, die beschichteten Gerichte zu trocknen oder zu waschen.</li>
	</ol>
</li>
	<li>Embryonale Stammzellen der Maus (A2lox und 129) Kultur<ol>
<li>Inkubieren Sie mESCs (A2lox und 129) Zellen in den 0,1% gelatinebeschichteten 60 mm Zellkulturschalen in mESC Wachstumsmedium bei 37 °C in einem 5% CO2-Inkubator. Das mESC-Wachstumsmedium besteht aus 85% Knock-out DMEM/F12, 15% Knock-out Serumersatz (KSR), 0,1 mM β-Mercaptoethanol (2ME), 2 mM GlutaMAX, 1% nicht-essentielle Aminosäure (NEAA), 1% Penicillin/Streptomycin (P/S), 1000 U/mL Leukämie-Hemmfaktor (LIF), 10 nM CHIR-99021 (GSK-3-Hemmer) und 0,33 nM PD0325901 (MEK-Hemmer). VORSICHT: β-Mercaptoethanol ist entzündlich und hat Inhalationstoxizität. Halten Sie sich von Brandquellen fern und tragen Sie eine Maske, um einEinatmen bei Gebrauch zu vermeiden.</li>
		<li>Ändern Sie das mESC-Wachstumsmedium täglich für ein besseres Wachstum von A2lox und 129.</li>
		<li>Wenn die Zellen 80% Zusammenfluss erreichen, entfernen Sie das Medium und fügen Sie 1 ml 0,1% Trypsin in die Schale. Sanft für 30 s schaukeln, um eine gleichmäßige Abdeckung von Trypsin auf allen Zellen zu gewährleisten.</li>
		<li>Lassen Sie die Zellen für etwa 1 min zu trypsinizeund dann entfernen Sie das Trypsin mit 1 ml Pipette.</li>
		<li>Fügen Sie 2 ml mL mESC Wachstumsmedium auf die Schale, Pipette nach oben und unten mehrmals, um eine einzellige Suspension zu machen.</li>
		<li>Zählen Sie die Dichte der Zellen in der Suspension so genau wie möglich mit Hämozytometer.</li>
		<li>Teilen Sie die Zellen in 7 Gruppen auf und induzieren Sie eine Differenzierung anhand verschiedener Protokolle, die in Tabelle 1dargestellt sind.</li>
	</ol>
</li>
</ol>2. Differenzierung von mESCs zu NPCs (Phase I)
<ol><li>0,1% gelatinebeschichtete Zellkulturplatten oder Decklippen zubereiten.<ol>
<li>Vor Gebrauch 0,1% gelatinebeschichtete 6-Well-Platten oder Decklippen wie in Schritt 1.1 zubereiten.</li>
	</ol></li>
	<li>Phase-I-Differenzierung mit Protokoll 1 (Tabelle 1)<ol>
<li>Samen ca. 2 x 104 mESCs in 2 ml Basaldifferenzierungsmedium I pro Brunnen in den 0,1% gelatinebeschichteten 6-Well-Platten. Überprüfen Sie die Zelldichte unter dem Mikroskop.</li>
		<li>Inkubieren Sie die Zellen bei 37 °C in einem 5% CO2-Inkubator für 6 h, um eine Anhaftung zu ermöglichen.</li>
		<li>Nach der Anhaftung nehmen Sie die Zellen aus dem Inkubator heraus und waschen Sie die Zellen zweimal mit 2 ml PBS.</li>
		<li>Fügen Sie jedem Brunnen 2 ml Basaldifferenzierungsmedium I (Tabelle 1) hinzu und legen Sie die Zellen wieder in den Inkubator.</li>
		<li>Lassen Sie die Zellen für 8 Tage zur Differenzierung. Ersetzen Sie das Basaldifferenzierungsmedium I alle 2 Tage.</li>
	</ol></li>
	<li>Phase-I-Differenzierung mit Protokoll 2 (Tabelle 1)<ol>
<li>Fügen Sie 1,5 x 106 mESCs in eine nicht klebende bakterielle Schale in 10 ml Basaldifferenzierungsmedium I ein, um die embryoide Körperbildung bei 37 °C in einem 5%CO2-Inkubator zu ermöglichen.</li>
		<li>Nach 2 Tagen zellische Aggregate in 15 ml Zentrifugenrohre übertragen und durch Schwerkraft absetzen lassen.</li>
		<li>Entfernen Sie den Überstand und fügen Sie 10 ml frisches Basaldifferenzierungsmedium I hinzu, um die embryoiden Körper wieder auszusetzen. Pflanzen Sie sie in eine neue nicht klebende bakterielle Schale ein und ermöglichen eine Differenzierung für weitere 2 Tage.</li>
		<li>Überprüfen Sie die Bildung von embryoiden Körpern unter dem Mikroskop (Abbildung 2A).</li>
		<li>Sammeln Sie embryoide Körper, wie in den Schritten 2.3.2-2.3.3 beschrieben. Samen etwa 50 embryoide Körper in 2 ml Basaldifferenzierungsmedium I pro Brunnen auf 0,1% gelatinebeschichtete 6-Well-Platten.</li>
		<li>1 mM All-Trans RA-Lager (in DMSO) vorbereiten und nach der Unterverpackung in einem -80 °C-Gefrierschrank vor dem Licht lagern. HINWEIS: RA ist instabil, und es sollte darauf geachtet werden, es von Licht fernzuhalten und den Luftkontakt während der Vorbereitung des RA-Bestands zu reduzieren.</li>
		<li>Für die RA-Induktion fügen Sie in jedem Bohrwert 2 L RA-Lager hinzu, um eine Endkonzentration von 1 M zu erreichen.</li>
		<li>Legen Sie die Platte bei 37 °C in den 5% CO2-Inkubator und differenzieren Sie sie weitere 4 Tage.</li>
		<li>Ändern Sie alle 2 Tage das gesamte 2 ml Basaldifferenzierungsmedium I (mit 1 M RA).</li>
	</ol></li>
	<li>Phase-I-Differenzierung mit Protokoll 3 (Tabelle 1)<ol>
<li>1,5 x 106 mESCs in eine nicht klebende bakterielle Schale in 10 ml Basaldifferenzierungsmedium I pflanzen. 2 Tage für die embryoide Körperbildung bei 37 °C in einem 5%CO2-Inkubator lassen.</li>
		<li>Überprüfen Sie die Bildung von embryoiden Körpern unter dem Mikroskop (Abbildung 2B).</li>
		<li>Zellaggregate in 15 ml Zentrifugenrohre übertragen und durch Schwerkraft absetzen lassen.</li>
		<li>Entfernen Sie den Überstand sorgfältig und fügen Sie 10 ml frisches Basaldifferenzierungsmedium I hinzu, um sie wieder auszusetzen.</li>
		<li>Säen Sie etwa 50 embryoide Körper in 2 ml Basaldifferenzierungsmedium I pro Brunnen auf 0,1% gelatinebeschichtete 6-Well-Platten.</li>
		<li>Für die RA-Induktion fügen Sie in jedem Bohrwert 2 L RA-Lager hinzu, um eine Endkonzentration von 1 M zu erreichen.</li>
		<li>Legen Sie die Platte bei 37 °C in den 5% CO2-Inkubator und differenzieren Sie sie für weitere 6 Tage. Ändern Sie alle 2 Tage das gesamte Basaldifferenzierungsmedium I (mit 1 M RA).</li>
	</ol></li>
	<li>Phase-I-Differenzierung mit Protokoll 4 (Tabelle 1)<ol>
<li>Samen ca. 2 x 104 mESCs innerhalb von 2 ml Basaldifferenzierungsmedium I pro Brunnen auf die 0,1% gelatinebeschichteten 6-Well-Platten. Legen Sie die Platte bei 37 °C in den 5% CO2-Inkubator, um eine Befestigung für 6 h zu ermöglichen.</li>
		<li>Nach der Befestigung die Zellen zweimal mit 2 ml PBS waschen. Fügen Sie jeweils 2 ml Basaldifferenzierungsmedium I zu jedem Brunnen hinzu und lassen Sie eine Differenzierung für 4 Tage im 5% CO2-Inkubator bei 37 °C zu.</li>
		<li>Für die RA-Induktion fügen Sie jedem Bohrgut 2 L All-Trans-RA-Bestand (die Arbeitskonzentration beträgt 1 M) hinzu, um weitere 4 Tage differenzierungzuregen.</li>
		<li>Ersetzen Sie im gesamten Prozess alle 2 Tage das gesamte Medium.</li>
	</ol></li>
	<li>Phase-I-Differenzierung mit Protokoll 5 (Tabelle 1)<ol>
<li>Samen ca. 2 x 104 mESCs innerhalb von 2 ml Basaldifferenzierungsmedium I pro Brunnen auf die 0,1% gelatinebeschichteten 6-Well-Platten. Legen Sie die Platte bei 37 °C in den 5% CO2-Inkubator, um eine Befestigung für 6 h zu ermöglichen.</li>
		<li>Waschen Sie die Zellen zweimal mit 2 ml PBS. Fügen Sie jeweils 2 ml Basaldifferenzierungsmedium I zu jedem Brunnen hinzu und lassen Sie eine Differenzierung für 2 Tage im 5% CO2-Inkubator bei 37 °C zu.</li>
		<li>Für die anschließende RA-Induktion fügen Sie jedem Bohrkörper 2 L RA-Bestand hinzu, um eine Endkonzentration von 1 m zu erreichen. Legen Sie die Platte bei 37 °C in den 5% CO2-Inkubator, um für weitere 6 Tage eine Differenzierung zu induzieren.</li>
		<li>Ersetzen Sie im gesamten Prozess alle 2 Tage das gesamte Medium.</li>
	</ol></li>
	<li>Phase-I-Differenzierung mit Protokoll 6 (Tabelle 1)<ol>
<li>1,5 x 106 mESCs in eine nicht klebende bakterielle Schale in 10 ml N2B27 Medium II(Tabelle 1) pflanzen, um die Bildung embryoider Körper zu ermöglichen.</li>
		<li>Sammeln Sie am2. Tag die in den Schritten 2.3.2-2.3.3 beschriebenen Zellaggregate und setzen Sie die embryoiden Körper mit 10 ml frischem N2B27-Medium II wieder aus.</li>
		<li>Pflanzen Sie sie in eine neue nicht klebende bakterielle Schale ein und lassen Sie sie für weitere 2 Tage im 5% CO2-Inkubator bei 37 °C differenzierungen. Überprüfen Sie die Bildung von embryoiden Körpern unter dem Mikroskop.</li>
		<li>Sammeln Sie am4. Tag embryoide Körper. Samen etwa 50 embryoide Körper pro Brunnen auf 0,1% gelatinebeschichtete 6-Well-Platten mit 2 ml N2B27 Medium II.</li>
		<li>Fügen Sie in jedem Brunnen 2 L All-Trans-RA-Bestände hinzu und induzieren Sie weitere 4 Tage Differenzierung. Ersetzen Sie das gesamte Medium (N2B27 medium II mit 1 M RA) alle zwei Tage.</li>
	</ol></li>
	<li>Phase-I-Differenzierung mit Protokoll 7 (Tabelle 1)<ol>
<li>Samen ca. 2 x 104 mESCs innerhalb von 2 ml Basaldifferenzierungsmedium I pro Brunnen auf die 0,1% gelatinebeschichteten 6-Well-Platten. Legen Sie die Platte bei 37 °C in den 5% CO2-Inkubator, um eine Befestigung für 6 h zu ermöglichen.</li>
		<li>Waschen Sie die Zellen zweimal mit PBS. Dann fügen Sie 2 ml N2B27 medium II zu jedem Brunnen hinzu und lassen Sie eine Differenzierung für 8 Tage bei 37 °C in einem 5% CO2-Inkubator zu.</li>
		<li>Ändern Sie das gesamte N2B27 Medium II alle 2 Tage.</li>
	</ol></li>
</ol>3. Zellmorphologie Beobachtung
<ol><li>Überprüfen Sie den Differenzierungsstatus der oben genannten 7 Gruppen täglich unter einem invertierten Phasenkontrastlichtmikroskop.</li>
	<li>Wählen Sie nach dem Zufallsprinzip mindestens 12 Felder aus, und nehmen Sie Fotos auf, um die morphologischen Veränderungen jeder Gruppe auf D8 aufzuzeichnen.</li>
</ol>4. Immunfluoreszenzfärbung
<ol><li>Probenvorbereitung: Seed mESCs auf 0,1% gelatinebeschichteten Abdeckungen und ermöglichen eine Differenzierung für 8 Tage unter Verwendung der in Schritt 2 genannten Protokolle.</li>
	<li>Spülen: Am8. Tag die Proben aus dem Inkubator nehmen und das Differenzierungsmedium durch Aspiration entfernen. Spülen Sie die Zellen einmal mit 1 ml PBS für 5 min.</li>
	<li>Fixierung: Fügen Sie 1 ml 4% Paraformaldehyd zu jeder Probe hinzu und fixieren Sie die Zellen für 20 min bei Raumtemperatur (RT).</li>
	<li>Spülen: Spülen Sie die Zellen nach der Fixierung mit 1 ml PBS 3 mal, jeweils 5 min.</li>
	<li>Permeabilisierung: Fügen Sie 1 ml 0,2% TritonX-100 in PBS zu jeder Probe hinzu und lassen Sie sie 8 min bei RT.</li>
	<li>Spülen: Spülen Sie die Zellen nach permeabilisieren der Zellen mit 1 ml PBS 3 mal, jeweils 5 min.</li>
	<li>Blockierung: Fügen Sie 1 ml 10% Ziegenserum in PBS zu jeder Probe hinzu und inkubieren Sie bei RT für 1 h, um alle unspezifischen Wechselwirkungen zu blockieren.</li>
	<li>Inkubation mit primärem Antikörper<ol>
<li>Verdünnen Sie den Anti-Nestin-Antikörper im Verhältnis 1:100 mit 5% Ziegenserum in PBS.</li>
		<li>500 l verdünnter Antikörper auf verschiedene Proben auftragen und über Nacht bei 4 °C inkubieren.</li>
	</ol>
</li>
	<li>Spülen: Entfernen Sie den Antikörper und spülen Sie die Proben vorsichtig mit 1 ml PBS 3 mal für jeweils 8 min.</li>
	<li>Inkubation mit Sekundärantikörper<ol>
<li>Verdünnen Sie das Alexa Fluor 488-markierte Ziegenanti-Maus-IgG im Verhältnis 1:500 mit 5% Ziegenserum in PBS.</li>
		<li>500 l verdünnter Antikörper auf verschiedene Proben auftragen und bei RT 2 h im Dunkeln brüten. HINWEIS: Führen Sie nach dem Auftragen eines fluoreszierenden Sekundärantikörpers alle nachfolgenden Schritte im Dunkeln aus, um eine Fluoreszenzabschreckung zu verhindern.</li>
	</ol>
</li>
	<li>Spülen: Entfernen Sie den sekundären Antikörper und spülen Sie die Proben vorsichtig mit 1 ml PBS 3 mal für jeweils 8 min.</li>
	<li>Nukleare Färbung und Montage<ol>
<li>Legen Sie einen Tropfen DAPI-Montagemedium auf den sauberen Mikroschlitten.</li>
		<li>Nehmen Sie die Proben vorsichtig von den Platten ab und legen Sie die Probe mit der Zelle nach unten auf das DAPI-Montagemedium. Lassen Sie im Dunkeln für 5 min bei RT.</li>
		<li>Entfernen Sie überschüssiges DAPI-Montagemedium mit saugfähigem Papier.</li>
	</ol>
</li>
	<li>Fluoreszenzmikroskopische Beobachtung<ol>
<li>Stellen Sie die Proben unter die Fluoreszenzmikroskopie und erkennen Sie das Signal für DAPI und Alexa Fluor 488 mit geeigneten Filtern.</li>
		<li>Wählen Sie gleichmäßig und zufällig 10-15 verschiedene visuelle Felder für jedes Sample aus und zeichnen Sie die Bilder mit einer CCD-Kamera auf.</li>
	</ol>
</li>
</ol>5. Differenzierung von NPCs zu Neuronen (Phase II)
<ol><li>Bereiten Sie mESC-Derivate nach Phase-I-Differenzierung nach Protokoll 3 (8 Tage, Tabelle 1) vor, wie in Schritt 2.4 beschrieben, das die höchste Differenzierungseffizienz hat (siehe Abbildung 3). HINWEIS: Nach der Phase-I-Differenzierung sollte die Qualitätskontrolle mit Hilfe der oben genannten Zellmorphologiebeobachtung und Immunfluoreszenz-Assay durchgeführt werden, um eine gesunde und ertragreiche NPCs zu gewährleisten.</li>
	<li>Samen ca. 5 x 105 mESC-Derivate innerhalb von 2 ml Basaldifferenzierungsmedium I pro Brunnen auf die 0,1% gelatinebeschichteten 6-Well-Platten. Teilen Sie die mESC-Derivate nach dem Zufallsprinzip in 3 Gruppen auf, wie Phase II-Protokoll 1, Protokoll 2 und Protokoll 3.</li>
	<li>Legen Sie die Platte bei 37 °C in den 5% CO2-Inkubator, um eine Befestigung für 6 h zu ermöglichen. Waschen Sie sie zweimal mit 2 ml PBS.</li>
	<li>Fügen Sie 2 ml Basaldifferenzierungsmedium I, N2B27 Medium I und N2B27 Medium II (Tabelle 2) zu jedem Brunnen der oben genannten Gruppen hinzu.</li>
	<li>Legen Sie die Platten in den Inkubator und lassen Sie für weitere 10 Tage unterscheiden. Ändern Sie das entsprechende Medium alle 2 Tage.</li>
	<li>Überprüfen Sie den Differenzierungsstatus und zeichnen Sie die morphologischen Veränderungen auf, wie in Schritt 3 erwähnt.</li>
	<li>Bewerten Sie am 18. Tag die Erzeugung von Neuronen (β-Tubulin III positiv) und bestimmen Sie die Differenzierungseffizienz der 3 Protokolle unter Verwendung von Schritt 4.</li>
</ol>

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F., 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=All-trans+retinoic+acid+promotes+neural+lineage+entry+by+pluripotent+embryonic+stem+cells+via+multiple+pathways.">All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways.</a> BMC Cell Biology. 10, 57 (2009).</li><li>Kanungo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+in+P19+stem+cell+differentiation.">Retinoic acid signaling in P19 stem cell differentiation.</a> Anticancer Agents in Medicinal Chemistry. 17 (9), 1184-1198 (2017).</li><li>Rochette-Egly, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+and+mouse+embryonic+stem+cell+differentiation:+Cross+talk+between+genomic+and+non-genomic+effects+of+RA.">Retinoic acid signaling and mouse embryonic stem cell differentiation: Cross talk between genomic and non-genomic effects of RA.</a> Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1851 (1), 66-75 (2015).</li><li>Wang, R., 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=Retinoic+acid+maintains+self-renewal+of+murine+embryonic+stem+cells+via+a+feedback+mechanism.">Retinoic acid maintains self-renewal of murine embryonic stem cells via a feedback mechanism.</a> Differentiation. 76 (9), 931-945 (2008).</li><li>Wu, H. B., 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=Retinoic+acid-induced+upregulation+of+miR-219+promotes+the+differentiation+of+embryonic+stem+cells+into+neural+cells.">Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells.</a> Cell Death Disease. 8, 2953 (2017).</li><li>Chen, W., 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=Retinoic+acid+regulates+germ+cell+differentiation+in+mouse+embryonic+stem+cells+through+a+Smad-dependent+pathway.">Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway.</a> Biochemical and Biophysical Research Communications. 418 (3), 571-577 (2012).</li><li>Nakayama, Y., 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+rapid+and+efficient+method+for+neuronal+induction+of+the+P19+embryonic+carcinoma+cell+line.">A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line.</a> Journal of Neuroscience Methods. 227, 100-106 (2014).</li><li>Bibel, 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=Differentiation+of+mouse+embryonic+stem+cells+into+a+defined+neuronal+lineage.">Differentiation of mouse embryonic stem cells into a defined neuronal lineage.</a> Nature neuroscience. 7 (9), 1003-1009 (2004).</li><li>Coyle, D. E., Li, J., Baccei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Differentiation+of+Retinoic+Acid-Induced+Human+Pluripotent+Embryonic+Carcinoma+Stem+Cell+Neurons.">Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons.</a> PLoS ONE. 6 (1), 16174 (2011).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Developmental Biology. 275, 124-142 (2004).</li></ol>

Die Autoren haben nichts zu verraten.

In der vorliegenden Studie haben wir eine einfache und effektive Methode zur neuronalen Differenzierung von mESCs mit kostengünstigen und leicht zu erhaltenden Materialien etabliert. Bei dieser Methode können 2 Tage embryoiden Körperbildung gefolgt von 6 Tagen RA-Induktion die Differenzierung von mESCs in NPCs wirksam fördern (Phase I-Protokoll 3). Für die Phase-II-Differenzierung induzieren N2B27 Medium II (Phase II-Protokoll 3) am effektivsten die Differenzierung von NPCs in Neuronen. Um den Erfolg zu gewährleist...

Embryonale Stammzellen (ESCs) sind pluripotent und können sich unter bestimmten Bedingungen in neuronale Vorläuferzellen (NPCs) und anschließend in Neuronen differenzieren1. ESC-basierte Neurogenese bietet die beste Plattform, um Neurogenese zu imitieren, und dient somit als nützliches Werkzeug für Entwicklungsbiologie-Studien und potenziell Hilfe in der regenerativen Medizin2,3. In den letzten Jahrzehnten wurden viele Strategien zur Induktion der embryonalen Neurogenese berichtet, wie die transgene Methode4, mit kleinen Molekülen5, mit einer 3D-Matrix-Mikroumgebung6, und die Co-Kultur-Technik7. Die meisten dieser Protokolle sind jedoch entweder bedingt oder schwer zu bedienen, daher sind sie für den Einsatz in den meisten Laboratorien nicht geeignet.
Um eine einfach zu bedienende und kostengünstige Methode zu finden, um eine effiziente neuronale Differenzierung von mESCs zu erreichen, wurde hier eine kombinatorische Screening-Strategie verwendet. Wie in Abbildung 1beschrieben, wurde der gesamte Prozess der embryonalen Neurogenese in 2 Phasen unterteilt. Phase I bezieht sich auf den Differenzierungsprozess von mESCs in NPCs, und Phase II bezieht sich auf die nachfolgende Differenzierung von NPCs in Neuronen. Basierend auf den Prinzipien der einfachen Bedienung, der niedrigen Kosten, der leicht zugänglichen Materialien und der hohen Differenzierungseffizienz wurden sieben Protokolle in Phase I und drei Protokolle in Phase II auf der Grundlage des traditionellen stickigen Monolayer-Kultursystems oder embryoiden Körperbildungssystems8,9ausgewählt. Die Differenzierungseffizienz von Protokollen in beiden Phasen wurde mittels Zellmorphologiebeobachtung und Immunfluoreszenz-Assay evaluiert. Durch die Kombination des effizientesten Protokolls jeder Phase haben wir die optimierte Methode zur neuronalen Differenzierung von mESCs etabliert.

<table>
	<tbody>
		<tr>
			<td>Anti-Nestin antibody [Rat-401]</td>
			<td>Abcam</td>
			<td>Ab11306</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III antibody produced in rabbit</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-Labeled Goat Anti-Mouse IgG</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>stored at -20 &#176;C, and protect from light</td>
		</tr>
		<tr>
			<td>CHIR-99021 (CT99021)</td>
			<td>Selleck</td>
			<td>S1263</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-Labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0516</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>DME/F-12 1:1 (1x)</td>
			<td>HyClone</td>
			<td>SH30023.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Gibco</td>
			<td>CM0635B</td>
			<td>stored at room temperature</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Immunol Staining Primary Antibody dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor human</td>
			<td>Sigma</td>
			<td>L5283</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI - Aqueous, Fluoroshield</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson</td>
			<td>005-000-121</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nonadhesive bacterial dish</td>
			<td>Corning</td>
			<td>3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin Solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PD0325901(Mirdametinib)</td>
			<td>Selleck</td>
			<td>S1036</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>stored at -80 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Strain 129 Mouse Embryonic Stem Cells</td>
			<td>Cyagen</td>
			<td>MUAES-01001</td>
			<td>Maintained in feeder-free culture system</td>
		</tr>
		<tr>
			<td>Stem-Cellbanker (DMSO free)</td>
			<td>ZENOAQ</td>
			<td>stem cellbanker DMSO free</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1X) Solution</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>6 - well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>NUNC</td>
			<td>339650</td>
			<td></td>
		</tr>
	</tbody>
</table>

neuronal differentiation from mouse embryonic stem cells in vitro

Die neuronale Differenzierung embryonaler Stammzellen (mESCs) ist ein mögliches Werkzeug zur Aufklärung der Schlüsselmechanismen der Neurogenese und potenzieller Hilfe in der regenerativen Medizin. Hier haben wir eine effiziente und kostengünstige Methode zur neuronalen Differenzierung von mESCs in vitroetabliert, mit der Strategie des kombinatorischen Screenings. Unter den hier definierten Bedingungen ermöglicht die 2-tägige embryoide Körperbildung + 6-Tage-Retinoinsäure-Induktionsprotokoll eine schnelle und effiziente Differenzierung von mESCs in neuronale Vorläuferzellen (NPCs), wie die Bildung von gut gestapelten und neuritigen A2lox- und 129 Derivaten sieht, die Nestin-positiv sind. Der gesunde Zustand von embryoiden Körpern und der Zeitpunkt, an dem Retinsäure (RA) angewendet wird, sowie die RA-Konzentrationen sind dabei von entscheidender Bedeutung. In der anschließenden Differenzierung von NPCs in Neuronen könnte N2B27 medium II (ergänzt durch Neurobasal medium) die langfristige Erhaltung und Reifung neuronaler Zellen besser unterstützen. Die vorgestellte Methode ist hochgradig effizient, kostengünstig und einfach zu bedienen und kann ein leistungsfähiges Werkzeug für die Neurobiologie und Entwicklungsbiologieforschung sein.

2-Tage embryoide Körperbildung + 6-Tage-RA-Induktion funktioniert am besten bei der Unterscheidung von mESCs in NPCs (Phase I). Um das optimale Protokoll zu bestimmen, das die Differenzierung von mESCs in NPCs (Phase I) am besten fördert, wurden 7 Protokolle sowohl auf A2lox als auch auf 129 mESCs (Tabelle 1) getestet und der Differenzierungsstatus jeder Gruppe mit Lichtmikroskop überwacht. Wie in Abbildung 3Adargestellt, zeigten die meisten A2lox- und 129-Derivate unter ...

Watch this Scientific Journal Video about Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro at JoVE.com

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Hier haben wir eine kostengünstige und einfach zu bedienende Methode etabliert, die eine schnelle und effiziente Differenzierung von embryonalen Stammzellen in Neuronen leitet. Diese Methode eignet sich für die Popularisierung unter Laboratorien und kann ein nützliches Werkzeug für die neurologische Forschung sein.

Neuronale Differenzierung von Maus embryonalen Stammzellen In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

neuronal-differentiation-from-mouse-embryonic-stem-cells-in-vitro

Research

JoVE Journal

Neuroscience

8.7K Aufrufe.  Wuhan Center for Disease Control and Prevention. Hier haben wir eine kostengünstige und einfach zu bedienende Methode etabliert, die eine schnelle und effiziente Differenzierung von embryonalen Stammzellen in Neuronen leitet. Diese Methode eignet sich für die Popularisierung unter Laboratorien und kann ein nützliches Werkzeug für die neurologische Forschung sein.

Serie: Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Effiziente Ableitung von humanen neuronalen Vorläuferzellen und Neuronen aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Forschung

Neurowissenschaften

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotypischen Kulturen von embryonalen ventralen Mittelhirn: Ein System zur Dopaminerge Neuronale Development Study In-vitro-</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, 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.

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 ...

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.

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 ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

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 ...

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.

Isolation und Kultur der embryonalen Maus neurale Stammzellen

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In Vitro Wedge Slice Vorbereitung für Diemimitieren In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

In-vitro-Modellierung für neurologische Erkrankungen mit direkter Konvertierung von Fibroblasten in neuronale Progenitorzellen und Differenzierung in Astrozyten

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging in Maus Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Humane Mikroglia-ähnliche Zellen: Differenzierung aus induzierten pluripotenten Stammzellen und In-vitro-Lebendzell-Phagozytose-Assay mit humanen Synaptosomen

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...