Wir danken K. Deisseroth und S. Je nach den ChR2 und Synapsin1-tdTomato lentiviralen Konstrukte bzw. C. Chai für den Austausch von Know-how und das Protokoll auf iPSC Generation, WY Leong für die technische Unterstützung, die Mitglieder der Goh-Labor für den Austausch von Reagenzien und Know-how. Diese Arbeit wurde von Abbott Nutrition and Academic Centre of Excellence (ACE) Forschungspreis von Glaxosmithkline (GSK) zu ELG im Rahmen ihrer wettbewerbsfähiges Forschungsprogramm (NRF 2008 NRF-CRP 002-082) zu ELG, die von der National Research Foundation Singapur und GJA und von der Weltklasse Institute (WCI) Programm der National Research Foundation of Korea (NRF), die vom Ministerium für Bildung, Wissenschaft und Technologie Korea (MEST) gefördert (NRF Förderkennzeichen: WCI 2009-003) zu GJA

<ol>
	<li>Wang, H., 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=High-speed+mapping+of+synaptic+connectivity+using+photostimulation+in+Channelrhodopsin-2+transgenic+mice.">High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice.</a> Proceedings of the National Academy of Sciences. 104 (19), 8143-8148 (2007).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale genetically targeted optical control of neural activity.</a> Nature Neuroscience. 8 (9), 1263-1268 (2005).</li><li>Nagel, 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proceedings of the National Academy of Sciences. 100 (24), 13940-13945 (2003).</li><li>Zhang, 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=Multimodal+fast+optical+interrogation+of+neural+circuitry.">Multimodal fast optical interrogation of neural circuitry.</a> Nature. 446 (7136), 633-639 (2007).</li><li>Stroh, 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=Tracking+stem+cell+differentiation+in+the+setting+of+automated+optogenetic+stimulation.">Tracking stem cell differentiation in the setting of automated optogenetic stimulation.</a> Stem Cells. 29 (1), 78-88 (2011).</li><li>Weick, J. P., Liu, Y., Zhang, S. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+neurons+adopt+and+regulate+the+activity+of+an+established+neural+network.">Human embryonic stem cell-derived neurons adopt and regulate the activity of an established neural network.</a> Proceedings of the National Academy of Sciences. 108 (50), 20189-20194 (2011).</li><li>Viviani, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+coculture+of+neurons+and+glial+cells.">Preparation and coculture of neurons and glial cells.</a> Curr Protoc Cell Biol. 32, 2.7.1-2.7.2.1 (2006).</li><li>Braun, H., G&#252;nther-Kern, A., Reymann, K., Onteniente, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+differentiation+of+human+iPS-cells+in+a+rat+cortical+primary+culture.">Neuronal differentiation of human iPS-cells in a rat cortical primary culture.</a> Acta Neurobiologiae Experimentalis. 72 (30), 219-229 (2012).</li><li>Shivaraj, M. C., 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=Taurine+induces+proliferation+of+neural+stem+cells+and+synapse+development+in+the+developing+mouse+brain.">Taurine induces proliferation of neural stem cells and synapse development in the developing mouse brain.</a> PLoS ONE. 7, e42935 (2012).</li><li>Okita, 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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nature Methods. 8 (5), 409-412 (2011).</li><li>Li, 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=Rapid+induction+and+long-term+self-renewal+of+primitive+neural+precursors+from+human+embryonic+stem+cells+by+small+molecule+inhibitors.">Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors.</a> Proceedings of the National Academy of Sciences. 108 (20), 8299-8304 (2011).</li><li>Chen, 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=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nature Methods. 8 (50), 424-429 (2011).</li><li>Packer, A. M., Roska, B., Hausser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+neurons+and+photons+for+optogenetics.">Targeting neurons and photons for optogenetics.</a> Nature Neuroscience. 16 (7), 805-815 (2013).</li><li>Marchetto, M. C. 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=A+Model+for+neural+development+and+treatment+of+Rett+Syndrome+using+human+induced+pluripotent+stem+cells.">A Model for neural development and treatment of Rett Syndrome using human induced pluripotent stem cells.</a> Cell. 143 (4), 527-539 (2010).</li><li>Asrican, 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=Next-generation+transgenic+mice+for+optogenetic+analysis+of+neural+circuits.">Next-generation transgenic mice for optogenetic analysis of neural circuits.</a> Frontiers in Neural Circuits. 7, 160 (2013).</li></ol>

No conflicts of interest declared.

Optogenetik stellt zeitliche und räumliche Präzision für die Aktivierung von definierten Populationen von Neuronen 13. In unseren Experimenten wurde der gesamte Bereich des Mikroskopobjektivs 30 Sek Photo stimulieren nur kortikalen Neuronen, ChR2 beleuchtet. Dies erlaubte uns, zu bestimmen, ob die synaptische Verbindungen wurden zwischen den verschiedenen Populationen von Neuronen in der Co-Kultur gebildet. Unsere Ergebnisse zeigen, dass Photo erhöht PSC Frequenz in iPS-abgeleitete Neuronen, was zeigt, dass diese Neuronen erhalten synaptischen Input von präsynaptischen Neurone Ausdruck ChR2. Dies wiederum festgestellt, dass die iPSC abgeleiteten Neuronen erfolgreich in Schaltungen mit den kortikalen Neuronen aufgenommen. Es gibt mehrere wichtige Schritte zur Erzeugung des Co-Kultursystem. Es ist wichtig, sicherzustellen, dass die Maus NPC abgeleiteten Astrocytenkulturen gesund sind, mit minimalem Zelltod, bevor mit Überzug ausgehendvon anderen Zelltypen. Kortikalen Neuronen und menschlichen NPCs und befestigen auf gesunde Astrozyten und elektrophysiologischen Ableitungen hängen stark von den Kulturbedingungen. Die andere wichtige Schritte in diesem Protokoll sind die Elektroporation und Plattieren von kortikalen Neuronen auf Astrozytenkulturen. Da eine große Anzahl von Zellen nach der Elektroporation sterben, ist es wichtig, sicherzustellen, dass zumindest 6 Millionen kortikalen Neuronen zum Ausplattieren auf Astrocytenkulturen in Platten mit 24 Vertiefungen elektroporiert. Wir haben beobachtet, dass, wenn weniger als 6 Millionen Zellen wurden elektroporiert, die Anzahl der Neuronen, die überlebt haben, nicht einen dichten neuronalen Netzwerk, das elektrophysiologische Aufzeichnungen betroffen bilden. Die Anzahl der durch Elektroporation kortikalen Neuronen sollte auch konsequent sein für jeden Co-Kultur-Experiment Vergleich zwischen unterschiedlichen Studien zu ermöglichen. Für alle Schritte, welche die enzymatische Verdauung, ist es wesentlich, die nicht an Zellen in Verdauungslösung zu lange, weil es zum Zelltod führen lassen. DieseAnsatz kann verwendet werden, um die Fähigkeit von Neuronen aus patientenspezifischen Fibroblasten synaptische Kontakte mit anderen Neuronen bilden screenen. Neuronen aus Fibroblasten von Patienten mit der Erkrankung des Nervensystems Rett-Syndrom (RTT) abgeleitet aufweisen synaptischen Übertragung Defekte: RTT Neuronen zeigen eine signifikante Abnahme der PSC Frequenz und Amplitude im Vergleich zu gesunden Neuronen, was vermutlich auf weniger synaptischer Verbindungen 14. Unsere Co-Kultursystem erlaubt die Untersuchung der Wirkung von Medikamenten auf Neuronen anzeigt Entwicklungsdefekte. Dies kann durch Zugabe von Verbindungen von Interesse bei der Aussaat von erkrankten menschlichen NSC sowie bei kontinuierlicher Exposition zu Verbindungen der ganzen Zeit der neuronalen Differenzierung durchgeführt werden. Während diese Co-Kultursystem kann auch als Modell für Drogentests verwendet werden, ist eine Einschränkung bei diesem Ansatz, dass der Prozess der Untersuchung der Auswirkungen der einzelnen Kandidatenverbindung kann lang und mühsam sein, wiees ist nicht ein Hochdurchsatz-Screening-Verfahren. Nach der Behandlung mit Wirkstoffkandidaten haben iPSC abgeleiteten Neuronen einzeln gepatcht werden, um die Auswirkungen der einzelnen Verbindungen auf PSCs zu bestimmen. Darüber hinaus gibt es noch nicht viele vorhanden Fibroblasten und iPS-Zellen von Patienten. Daher wird es von Interesse, in der nahen Zukunft sein, mehr Patientenzellen und auch auf dieses Protokoll für Hochdurchsatz-Screening angepasst werden. Zum Beispiel durch Markierung iPSC abgeleiteten Neuronen mit einem Indikator, wie beispielsweise genetisch codierten Sensoren von Ionen oder des Membranpotentials, wäre es möglich, die Durchlaufgeschwindigkeit im Wesentlichen durch Seitenschritt die zeitraubelektrophysiologische Prozedur zu erhöhen. Da der gesamte Vorgang der Erzeugung dieses Co-Kultursystem, von Umprogrammierung Fibroblasten zur Durchführung elektrophysiologischen Ableitungen, benötigt mehr als 90 Tage, ist es empfehlenswert, entweder die menschlichen iPSCs oder NSC erweitert werden, nach der Umprogrammierung und neurale Induktion Schritte jeweilsund kryokonserviert, um die Zeit für zukünftige Experimente nötig zu verringern. In unseren Experimenten haben wir ausgedrückt ChR2 in kortikalen Neuronen unter synapsin1 Promotors. Da dieser Promotor pan-neuronalen wir vermutlich wurden beide hemmenden und erregenden kortikalen Neuronen photostimulating. Wenn das Ziel der zukünftigen Experimenten ist, spezifisch abzufragen entweder inhibitorisch oder exzitatorischer Schaltungen könnten spezifischeren Promotoren verwendet werden. Alternativ könnte kortikalen Neuronen aus transgenen (oder Virus-injizierten Mäusen) wobei ChR2 Expression spezifisch genetisch definierte Subpopulationen von Neuronen gezielt erhalten werden. Zum Beispiel, Ernte kortikalen Gewebe von einer Maus, die Cre-Rekombinase hat ausgedrückt in Parvalbumin haltige Inter (PV Inter), die mit einer Maus mit floxed ChR2 gekreuzt ist eine Situation, in der nur kortikalen Neuronen, ChR2 wird PV Inter 15 werden erhalten. Die Verwendung solcher ChR2-exprimierenden Inter in unserer gleichzeitig culture System ermöglichen die Bestimmung, ob ein patched iPSC abgeleiteten Neuron ist in der Lage, in einen Stromkreis mit PV Inter integrieren. Basierend auf einer früheren Studie 9, sind kortikalen Neuronen reifen mit überlappenden Dendriten nach 14 Tagen in vitro. Wenn also die Photo keine Antwort von einem patched iPSC abgeleiteten Neuron hervorrufen sollte darauf hinweisen, dass das Neuron nicht die Fähigkeit, synaptische Verbindungen mit kortikalen Neuronen zu machen. Ein iPSC abgeleiteten Neuron kann synaptischen Eingang entweder von anderen iPSC abgeleiteten Neuronen oder kortikalen Neuronen empfangen. Wenn traditionelle Patch-Clamp-Messungen verwendet wurden, wäre es nicht möglich, zu unterscheiden, wo das synaptische Eingabe kommt. Der Vorteil unseres Systems ist die postsynaptische Reaktionen aus kortikalem präsynaptischen Eingang selektiv evozierten und identifiziert werden. Die hier beschriebenen Verfahren bieten einen einfachen Einstieg in die Fähigkeit der iPS-abgeleiteten Neuronen bewerten zu integrierenin einen definierten neuronalen Netzwerks.

In den letzten Jahren haben unser Verständnis des Neurons und der neuronalen Schaltungsfunktion durch mehrere optogenetische Werkzeuge, die neuronale Erregbarkeit steuern revolutioniert. Ein solches Instrument ist das Licht-aktivierten Kationen-Kanal, Channelrhodopsin-2 (ChR2): Neuronen, ChR2 Feuer Aktionspotentiale in Reaktion auf Lichtstimulation 1-3. Da Neuronen normalerweise nicht empfindlich auf Licht, öffnet sich diese bemerkenswerte Fähigkeit neue Wege für die genaue zeitliche und räumliche Steuerung der Aktivität von genetisch definierten Populationen von Neuronen 4 und hat sich stark beschleunigt Studien der Hirnfunktion. ChR2 wurde angewandt, um Zellforschung für verschiedene Zwecke ergeben, von der Überwachung der neuronalen Entwicklung 5 bis Regulierung Aktivität neuronaler Netzwerke 6. Hier ChR2 verwendet wir, um die Nützlichkeit eines neuronalen Cokultursystem erhöhen. Co-Kultur-Systeme ermöglichen die Bewertung von Interaktionen zwischen verschiedenen Zelltypen.Co-Kulturen von Neuronen und Gliazellen, die Hauptzelltypen des Nervensystems werden häufig verwendet, um zu untersuchen Signalisierung zwischen den verschiedenen Zelltypen, sowie die Bedeutung dieser Signalisierung für physiologische Reaktionen zu bewerten, die Ausbreitung des Schadens und die Studie molekularen Mechanismen, die möglicherweise in dieser Signal 7 beteiligt sind. Eine frühere Untersuchung zeigte, dass menschliche iPSCs unterscheiden sich sehr schnell in die Neuronen in Gegenwart von Rattenkortexprimärkultur enthält Neuronen, Astrozyten, Oligodendrozyten und Mikroglia 8. In diesem Protokoll zeigen wir eine Methode, die ChR2 nutzt in einer Co-Kultursystem zur synaptischen Verbindungen zwischen Ratte kortikalen Neuronen und Neuronen aus humanen induzierten pluripotenten Stammzellen (iPS-Zellen) abgeleitet verhören. Wir beschreiben Schritte zu zerlegen und zu ernten Hirngewebe 9 sowie zu pflegen und zu differenzieren Maus neurale Vorläuferzellen (NPCs) in Astrozyten und menschlichen NPCs into Neuronen, die Co-Kultursystem zu schaffen. Um diese Co-Kultursystem zu etablieren, haben wir die von Okita et al Integration freie Umprogrammierung Verfahren. 10 die menschliche iPS-Zellen zu erzeugen. Diese iPS-Zellen wurden dann effizient in NPCs in chemisch definierten Bedingungen differenziert mit niedermolekularen Inhibitoren wie von Li et al. 11 In den Co-Kulturen können die verschiedenen Populationen von Neuronen über Erkennung des ChR2 Expression in kortikale Neuronen und Markierung von iPSC abgeleiteten Neuronen über das fluoreszierende Protein, tandem-Dimer Tomate (tdTomato) identifiziert werden. Kombinieren elektrophysiologische Aufzeichnungen von iPSC abgeleiteten Neuronen mit optogenetische Photo der kortikalen Neuronen ermöglicht Einschätzung der Fähigkeit dieser zwei Arten von Neuronen zu einander bilden. Insbesondere Photo von ChR2-exprimierenden kortikalen Neuronen hervorrufen synaptischen Antworten in iPS-abgeleitete Neuronen, die synaptischen Schaltungen gebildet habenmit der photo kortikalen Neuronen-Netzwerk. Wir zeigen, dass erhöhte postsynaptische Ströme sind in der Tat in iPS-abgeleiteten Neuronen unter diesen Bedingungen erkannt werden. Dieses Protokoll ermöglicht die Beurteilung der synaptischen Schaltung unter einer Vielzahl von experimentellen Bedingungen einschließlich Rekapitulation verschiedenen neurologischen Erkrankungsmodellen unter Verwendung iPSCs von Fibroblasten aus Patienten mit Morbus abgeleitet.

an optogenetic approach for assessing formation of neuronal connections in a co-culture system

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

Hier wird ein Protokoll der mehreren Schritte beim Erzeugen einer Co-Kultur, die aus Astrozyten beteiligt beschreiben, kortikale Neuronen und humanen Neuronen dargestellt. Menschen iPSCs wurden durch Neuprogrammierung Fibroblasten mit episomalen erhalten Vektoren 10 und spezifiziert in ein neuronales Linie mit einem Cocktail aus niedermolekularen Inhibitoren, so dass NPCs 11, die bei hoher Dichte (1A) gehalten wurden. Mehrere Schritte sind nötig, um die Co-Kultur zu generieren: Differenzierung von Maus-NPCs in Astrozyten, Beschichtung der Ratte kortikalen Neuronen, ChR2, und Aussaat der menschlichen NPCs mit tdTomato (1B) markiert. An Tag 2 der Differenzierung, können saure Gliafaserprotein (GFAP) + Astrozyten ohne weiteres in unseren Kulturen (1C) zu beachten. Maus NSC wurden für mindestens eine Woche vor dem Beschichten unterscheiden kortikalen Neuronen, ChR2 in die Astrozyten Monoschicht (1D). Nach 3bis 4 Wochen menschlicher NPC Differenzierung wurden tdTomato + -Neuronen in Kulturen (1E) detektiert. Diese Co-Kultursystem ermöglicht die konsistente Erfassung der postsynaptischen Ströme von einzelnen iPSC abgeleiteten Neuronen in Reaktion auf Lichtstimulation (2A). Wenn sie durch Licht angeregt wurden Aktionspotentiale in kortikalen Neuronen, ChR2 (2B) erzeugt wird. iPSC abgeleiteten Neuronen auch erregbaren (2C), welche erhöhte neuronaler Aktivität als die Amplitude der depolarisierenden Stromimpulse erhöht wurde (2D). Damit diese Zellen reife neuronale Eigenschaften. In der Abwesenheit von Licht, empfangen iPSC abgeleiteten Neurone spontan synaptischen Eingaben (2E). Diese inneren postsynaptischen Ströme wurden überwiegend von AMPA-Rezeptoren vermittelt, da Ströme durch GABA-Rezeptoren würde nach außen und NMDA-Rezeptoren tragen keine aktuellen eint die Haltepotential von -70 mV. Mindestens einige dieser Eingänge den von präsynaptische kortikale Neuronen, ChR2, weil Lichtstimulation der Frequenz des postsynaptischen Ströme (2E) stark erhöht. Der Zeitverlauf der Erhöhung der synaptischen Eingang in 2F dargestellt: Photo der kortikalen Neuronen wurde während des 30 s langen Lichtblitz aufrechterhalten. Während die Frequenz der PSCs wurde erhöht, wenn ChR2-exprimierende Neurone wurden photostimulierte (Figur 2G), war ihre Amplitude unbeeinflusst (Figur 2H). Zusammenfassend zeigen diese Ergebnisse, dass die iPS-abgeleiteten Neuronen zeigte reife neuronale Eigenschaften und könnte synaptischen Verbindungen mit präsynaptischen Neurone zu bilden. <img alt="Figur 1" src="/files/ftp_upload/52408/52408fig1highres.jpg" /> Abbildung 1: Schematische Darstellung zur Erzeugung KokulturSystem. (A) Timeline zur Umprogrammierung menschlichen Fibroblasten in iPS-Zellen und neurale Induktion an NPCs. (B) Zeitleiste für die terminale Differenzierung der menschlichen NPCs zu reifen Neuronen auf kortikalen Neuronen und Astrozyten-Kulturen. (C) Maus NPCs schnell in GFAP unterscheiden + Astrozyten nach 2 Tagen in vitro. (D) nach einer Woche kortikalen Neuronen, ChR2 wurden auf GFAP + Astrozyten bei 4 bis 5 Wochen nach der Differenzierung menschlicher NSC plattiert. (E), wurden elektrophysiologische Aufzeichnungen auf tdTomato + Neuronen durchgeführt. Maßstabsbalken entspricht 100 &amp; mgr; m. <img alt="Abbildung 2" src="/files/ftp_upload/52408/52408fig2highres.jpg" /> Abbildung 2: optogenetische Analyse der funktionalen synaptischer Verbindungen. (A) iPS-abgeleiteten Zellen mit tdTomato (rot) markiert waren mit kortikalen Neuronen, co-kultiviert das lichtaktivierteKanal, ChR2 (grün). Aufnahmen aus iPS-abgeleiteten Neuronen zeigte postsynaptischen Strom (PSC) Antworten, die entweder von den kortikalen Neuronen oder andere iPSC abgeleiteten Neuronen kommen könnte. (B) Beleuchtung (blauer Balken) rief eine Reihe von Aktionspotentialen in kortikalen Neuronen, ChR2. ( C) iPS-abgeleiteten Zellen sind durch Strominjektion hervorgerufen. (D) Brenn vs Stromkurve von iPS-Zellen abgeleitet. (E) Photostimulation der ChR2-exprimierenden kortikalen Neuronen (blauer Balken) erhöht die Frequenz der PSCs in iPS-abgeleiteten Neuronen . (F) den zeitlichen Verlauf der Zunahme der PSC Frequenz durch Ausleuchtung erzeugt. Blaue Balken zeigt die Zeit der Photostimulation. (G, H) Während PSC Frequenz wurde durch Photo (G) erhöht wird, PSC Amplitude unverändert war (H). * Zeigt Ihnen, p &lt;0,05 im Vergleich zu mit Student-t-Test zu kontrollieren.

Watch this Scientific Journal Video about An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System at JoVE.com

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Ein optogenetische Ansatz zur Abschätzung Bildung neuronaler Verbindungen in einer Co-Kultursystem

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

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Developmental Biology

13.3K Aufrufe.  Duke-NUS Graduate Medical School. A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2. 

Serie: An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Kultur und Co-Kultur von Maus Eierstöcke und Eibläschen

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

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Entwicklungsbiologie

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Alginat Encapsulation pluripotenter Stammzellen Mit einer Co-Axial Düsen

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Präziser zellulärer Ablationsansatz zur Modellierung akuter Nierenverletzungen bei der Entwicklung von Zebrafisch

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Ein standardisierter Ansatz für Multispezies Reinigung von männlichen männlichen Keimzellen durch mechanische Gewebedissoziation und Durchflusszytometrie

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Die Aorta Ring Kokultur Assay: Ein komfortables Werkzeug, um angiogenen Potenzial des mesenchymaler Stromazellen Zellen In Vitro zu bewerten

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Ein In-vitro- Modell eines Systems Parallel-Platte Perfusion, bakteriellen Einhaltung der Gewebe Transplantat zu studieren

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Dreidimensionales Angiogenese-Assay-System mit Co-Culture-Spheroiden, die von endotheliaalen Koloniebildenden Zellen und mesenchymalen Stammzellen gebildet werden

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Eine Co-Kultur-Methode zur Untersuchung des Neuriten-Auswuchss als Reaktion auf Dental Pulp Paracrine Signale

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Ein optimiertes O9-1/Hydrogel-System zur Untersuchung mechanischer Signale in Neuralleistenzellen

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Lichtinduzierte GFP-Expression in Zebrafischembryonen mit dem optogenetischen TAEL/C120-System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...