Ringraziamo K. Deisseroth e S. Je per i costrutti lentivirali ChR2 e Synapsin1-tdTomato rispettivamente C. Chai per la condivisione di know-how e il protocollo sulla generazione iPSC, WY Leong per il supporto tecnico, i membri del laboratorio Goh per la condivisione di reagenti e competenze. Questo lavoro è stato sostenuto da Abbott Nutrition e il Centro Accademico di Eccellenza (ACE) premio di ricerca da GlaxoSmithKline (GSK) per ELG, dal National Research Foundation di Singapore sotto la sua Competitive Research Program (NRF 2008 NRF-CRP 002-082) a ELG e GJA, e dalla Classe World Institute (WCI) Programma della Fondazione di Ricerca Nazionale di Corea (NRF), finanziato dal Ministero dell&#39;Istruzione, della Scienza e della Tecnologia di Corea (MEST) (NRF Concessione numero: WCI 2009-003) a 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.

Optogenetics fornisce precisione spaziale e temporale per l&#39;attivazione di popolazioni di neuroni definiti 13. Nei nostri esperimenti, l&#39;intero campo dell&#39;obiettivo del microscopio è stato illuminato per 30 secondi per foto-stimolare solo neuroni corticali esprimono ChR2. Questo ci ha permesso di determinare se le connessioni sinaptiche sono formate tra le diverse popolazioni di neuroni all&#39;interno della co-coltura. I nostri risultati hanno rivelato che fotostimolazione aumenta la frequenza PSC in neuroni iPSC-derivati, il che dimostra che questi neuroni ricevono input sinaptico dei neuroni corticali presinaptici esprimono ChR2. Questo, a sua volta, ha stabilito che i neuroni iPSC-derivati ​​incorporati con successo nei circuiti con i neuroni corticali. Ci sono diversi passaggi critici per la generazione di questo sistema di co-coltura. È importante assicurare che le colture di astrociti topo NPC-derivati ​​sono sani, con morte cellulare minima, prima di procedere con la placcaturadi altri tipi di cellule. Neuroni corticali e NPC umani attaccano bene su astrociti sani e registrazioni elettrofisiologiche dipendono fortemente dalle condizioni di coltura. Gli altri passaggi fondamentali in questo protocollo sono l&#39;elettroporazione e la placcatura dei neuroni corticali in colture di astrociti. Come un gran numero di cellule muoiono dopo l&#39;elettroporazione, è importante assicurare che almeno 6 milioni di neuroni corticali sono elettroporate per la placcatura in colture astrociti in piastre da 24 pozzetti. Abbiamo osservato che quando sono state elettroporate meno di 6 milioni di cellule, il numero di neuroni che sopravvivono non formano una rete neuronale densa, che ha colpito registrazioni elettrofisiologiche. Il numero di neuroni corticali elettroporate dovrebbe essere coerente per ogni esperimento di co-coltura per consentire il confronto tra i diversi studi. Per tutte le iniziative che coinvolgono la digestione enzimatica, è fondamentale non lasciare le cellule in soluzione digerente troppo a lungo in quanto può portare alla morte cellulare. Questoapproccio può essere utilizzato per schermare la capacità di neuroni derivati ​​da fibroblasti specifici del paziente per formare contatti sinaptici con altri neuroni. I neuroni derivati ​​da fibroblasti di pazienti affetti da disturbo dello sviluppo neurologico Sindrome di Rett (RTT) presentano difetti di trasmissione sinaptica: neuroni RTT mostrano una significativa riduzione della frequenza PSC ed ampiezza rispetto a neuroni sani, presumibilmente a causa della minore connettività sinaptica 14. Il nostro sistema di co-coltura permette esame degli effetti dei farmaci sui neuroni che mostrano difetti di sviluppo. Questo può essere effettuato tramite l&#39;aggiunta di composti di interesse durante la semina di NPC umani malate nonché durante l&#39;esposizione continua a composti per tutto il tempo di differenziazione neuronale. Mentre questo sistema di co-coltura può anche essere utilizzato come modello per il test della droga, un limite di questo approccio è che il processo di studio degli effetti di ciascun composto candidato può essere lunga e noiosa comenon è un metodo di screening ad alto rendimento. Dopo il trattamento con i composti candidati, neuroni COPSI derivate devono essere patch individualmente per determinare gli effetti di ciascun composto in PSC. Inoltre, ci sono attualmente non sono molti fibroblasti disponibili e iPSCs di pazienti. Pertanto, sarà di interesse nel prossimo futuro di avere cellule più pazienti e anche di adattare questo protocollo per lo screening ad alta produttività. Ad esempio, etichettando neuroni COPSI-derivati ​​con un indicatore di attività, quali sensori geneticamente codificati di ioni o potenziale di membrana, sarebbe possibile aumentare la velocità di flusso sostanzialmente by-side passo della procedura elettrofisiologica richiede tempo. Come l&#39;intero processo di generazione di questo sistema di co-coltura, dalla riprogrammazione fibroblasti di eseguire registrazioni elettrofisiologiche, richiede più di 90 giorni, si raccomanda che sia le iPSCs o NPC umani essere ampliato, dopo la riprogrammazione e fasi di induzione neurale, rispettivamente,e crioconservati per ridurre il tempo necessario per futuri esperimenti. Nei nostri esperimenti, abbiamo espresso ChR2 in neuroni corticali sotto il promotore synapsin1. Perché questo promotore è pan-neuronale, siamo stati presumibilmente fotostimolante entrambi i neuroni corticali inibitorie ed eccitatorie. Se l&#39;obiettivo di esperimenti futuri è interrogare specificamente circuiti sia inibitori o eccitatori, potrebbero essere utilizzati promotori più specifici. In alternativa, i neuroni corticali potrebbero essere ottenuti da transgenici (o virus iniettato topi), dove l&#39;espressione ChR2 è specificamente mirato a sottopopolazioni geneticamente definiti di neuroni. Ad esempio, i tessuti corticali raccolta da un mouse che ha Cre ricombinasi espresse in interneuroni-parvalbumin contenente (interneuroni PV) che viene attraversato con un mouse con floxed ChR2 produrrà una situazione in cui gli unici neuroni corticali esprimenti ChR2 saranno interneuroni PV 15. L&#39;uso di tali ChR2 esprimono interneuroni nella nostra co-cuSistema lture consentirà determinare se una patch neurone iPSC-derivato è in grado di integrare in un circuito con interneuroni fotovoltaici. Sulla base di uno studio precedente 9, neuroni corticali sono maturi con dendriti sovrapposizione dopo 14 giorni in vitro. Così, se fotostimolazione non suscita una risposta da un neurone patch COPSI-derivata, esso dovrebbe indicare che il neurone non ha la capacità di effettuare connessioni sinaptiche con neuroni corticali. Un neurone iPSC-derivato è in grado di ricevere input sinaptico o da altri neuroni iPSC-derivati ​​o neuroni corticali. Se sono stati usati toppa tradizionale registrazioni clamp, non sarebbe possibile distinguere se l&#39;ingresso sinaptica proviene. Il vantaggio del nostro sistema è che le risposte post-sinaptici di ingresso presinaptica corticale possono essere selettivamente evocati e identificati. Le procedure descritte qui forniscono un approccio semplice per valutare la capacità dei neuroni COPSI derivate integrarein una rete neuronale definito.

Negli ultimi anni, la nostra comprensione del neurone e la funzione del circuito neuronale è stato rivoluzionato da diversi strumenti optogenetic che controllano l&#39;eccitabilità neuronale. Uno di questi strumenti è il canale cationico luce attivato, channelrhodopsin-2 (ChR2): i neuroni che esprimono ChR2 potenziali d&#39;azione il fuoco in risposta alla stimolazione luminosa 1-3. Perché i neuroni non sono normalmente sensibili alla luce, questa notevole capacità apre nuove strade per il controllo preciso temporale e spaziale delle attività delle popolazioni geneticamente definiti di neuroni 4 e ha notevolmente accelerato studi di funzione cerebrale. ChR2 è stata applicata a ricerca sulle cellule staminali per scopi diversi, dal monitoraggio dello sviluppo neuronale 5 a regolare l&#39;attività delle reti neurali 6. Qui abbiamo usato ChR2 per aumentare l&#39;utilità di un sistema di co-coltura neuronale. Sistemi di co-coltura consentono la valutazione delle interazioni tra diversi tipi di cellule.Co-colture di neuroni e glia, i principali tipi di cellule del sistema nervoso, sono comunemente usati per studiare segnalazione tra questi diversi tipi di cellule, come pure per valutare la significatività di questa segnalazione per le risposte fisiologiche, la propagazione del danno e per esaminare la meccanismi molecolari che sono potenzialmente coinvolti in questa segnalazione 7. Un precedente studio ha rivelato che iPSCs umani si differenziano molto rapidamente in neuroni in presenza di ratto coltura contenente neuroni corticali primarie, astrociti, oligodendrociti, microglia e 8. In questo protocollo, dimostriamo un metodo che utilizza ChR2 in un sistema di co-coltura di interrogare connessioni sinaptiche tra i neuroni corticali di ratto e neuroni derivati ​​da cellule staminali umane pluripotenti indotte (iPSCs). Descriviamo passi di sezionare e raccolto tessuti cerebrali 9, nonché per mantenere e differenziare le cellule progenitrici neurali topo (NPC) in astrociti e NPC umani INTo neuroni per creare il sistema di co-coltura. Per stabilire questo sistema di co-coltura, abbiamo utilizzato il metodo di riprogrammazione senza integrazione descritto da Okita et al. 10 per generare iPSCs umani. Questi iPSCs stati poi efficacemente differenziati in NPC in condizioni chimicamente definite utilizzando inibitori piccole molecole come descritto da Li et al. 11 In co-culture, le diverse popolazioni di neuroni possono essere identificate tramite il rilevamento di espressione ChR2 in neuroni corticali e tagging dei neuroni iPSC-derivati ​​tramite la proteina fluorescente, tandem dimero pomodoro (tdTomato). Combinando registrazioni elettrofisiologiche da neuroni COPSI-derivati ​​con fotostimolazione optogenetic dei neuroni corticali consente di valutare la capacità di questi due tipi di neuroni per formare circuiti con l&#39;altro. In particolare, fotostimolazione di ChR2 esprimono neuroni corticali evocare risposte sinaptiche nei neuroni iPSC-derivati ​​che si sono formati i circuiti sinapticicon la rete neurone corticale fotostimolata. Abbiamo dimostrato che l&#39;aumento delle correnti postsinaptiche sono infatti rilevati in neuroni iPSC-derivati ​​in tali condizioni. Questo protocollo permette di valutare circuiteria sinaptica sotto una varietà di condizioni sperimentali, inclusi ricapitolazione diversi modelli di malattie neurologiche utilizzando iPSCs derivati ​​da fibroblasti da pazienti con malattia.

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.

Qui, un protocollo che descrive i diversi passaggi necessari per generare una co-coltura costituito astrociti, neuroni corticali e neuroni umani è illustrato. IPSCs umani sono stati ottenuti riprogrammando fibroblasti utilizzando vettori episomiale 10 e specificati in un lineage neurale con un cocktail di inibitori di piccole molecole, rendendo NPC 11 che sono stati mantenuti ad alta densità (Figura 1A). Diversi passi sono necessari per generare il co-coltura: differenziazione dei NPC topo in astrociti, placcatura di ratto neuroni corticali esprimono ChR2, e semina di NPC umani taggati con tdTomato (Figura 1B). Al giorno 2 di differenziazione, gliale fibrillare acida Protein (GFAP) + astrociti possano essere prontamente rilevate nelle nostre culture (Figura 1C). Mouse NPC è stato permesso di differenziare per almeno una settimana prima di placcatura neuroni corticali esprimono ChR2 sul monostrato astrociti (Figura 1D). Dopo 3a 4 settimane di differenziazione NPC umana, tdTomato + neuroni sono stati individuati nelle culture (Figura 1E). Questo sistema di co-coltura consente il rilevamento coerente di correnti provenienti da singoli neuroni postsinaptici COPSI-derivati ​​in risposta alla stimolazione luminosa (Figura 2A). Quando stimolata dalla luce, potenziali d&#39;azione sono stati generati in neuroni corticali esprimono ChR2 (Figura 2B). iPSC-derivati ​​neuroni sono eccitabili (Figura 2C), che mostra un aumento del potenziale d&#39;azione di tiro come l&#39;ampiezza di impulsi di corrente depolarizzante è stato aumentato (Figura 2D). Così, queste cellule mature presentano proprietà neuronali. In assenza di luce, i neuroni iPSC-derivati ​​hanno ricevuto ingressi sinaptici spontanei (Figura 2E). Queste correnti verso l&#39;interno postsinaptiche sono stati prevalentemente mediati dai recettori AMPA, perché le correnti attraverso i recettori GABA sarebbe andata e recettori NMDA non portano corrente unt il potenziale di -70 mV in possesso. Almeno alcuni di questi ingressi erano di neuroni corticali presinaptici esprimono ChR2, perché stimolazione luminosa aumenta notevolmente la frequenza di correnti postsinaptici (figura 2E). La durata di questo aumento di ingresso sinaptica è mostrato in Figura 2F: fotostimolazione dei neuroni corticali è stato sostenuto tra 30 sec luce flash lungo. Mentre la frequenza di PSC era elevato quando ChR2 esprimenti neuroni corticali stati fotostimolata (figura 2G), l&#39;ampiezza è stata influenzata (Figura 2H). In sintesi, questi risultati indicano che i neuroni iPSC-derivati ​​esposti maturi proprietà neuronali e potrebbero formare connessioni sinaptiche con neuroni corticali presinaptici. <img alt="Figura 1" src="/files/ftp_upload/52408/52408fig1highres.jpg" /> Figura 1: diagrammi schematici per la generazione di co-colturasistema. (A) Timeline per riprogrammare fibroblasti umani in iPSCs e induzione neurale di NPC. (B) Timeline per la differenziazione terminale di NPC umani in neuroni maturi sui neuroni e astrociti culture corticali. (C) del mouse NPC rapidamente differenziarsi in GFAP + astrociti dopo 2 giorni in vitro. (D) Dopo una settimana, neuroni corticali esprimono ChR2 stati placcati su GFAP + astrociti. (E) Alle 4 a 5 settimane dopo la differenziazione di NPC umani, registrazioni elettrofisiologiche sono stati eseguiti su tdTomato + neuroni. Barre di scala rappresentano 100 micron. <img alt="Figura 2" src="/files/ftp_upload/52408/52408fig2highres.jpg" /> Figura 2: analisi optogenetic della connettività sinaptica funzionale. (A) iPSC-derivato cellule taggati con tdTomato (rosso) sono stati co-coltura con neuroni corticali che esprime la luce attivatoCanale, ChR2 (verde). Le registrazioni da neuroni iPSC-derivati ​​rivelato correnti (PSC) risposte post-sinaptici, che potrebbero venire da entrambi i neuroni corticali o altri neuroni iPSC-derivati. (B) Illuminazione (barra blu) evoca una serie di potenziali d&#39;azione nei neuroni corticali esprimono ChR2. ( cellule C) iPSC-derivati ​​sono evocati da iniezione di corrente. (D) Firing vs attuale curva di cellule iPSC-derivate. (E) Fotostimolazione delle ChR2 esprimono neuroni corticali (barra blu) sono aumentate la frequenza di PSC in neuroni iPSC-derivati . corso (F) Tempo di aumento della frequenza PSC prodotto da fotostimolazione. Blu barra indica il tempo di fotostimolazione. (G, H) mentre la frequenza PSC è stato incrementato di fotostimolazione (G), l&#39;ampiezza PSC è stata influenzata (H). * Indica p &lt;0,05 rispetto al controllo con t-test di Student.

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.

Un approccio per la valutazione optogenetic Formazione di connessioni neuronali in un sistema di co-coltura

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 Views.  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. 

Video: 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.

Cultura e co-coltura di mouse ovaie e follicoli ovarici

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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Biologia dello sviluppo

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.

Incapsulamento alginato di cellule staminali pluripotenti usando un ugello coassiale

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.

Approccio preciso di ablazione cellulare per la modellazione di lesioni acute del rene nello sviluppo di 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 ...

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.

Un approccio standardizzato per la depurazione multispecie di cellule germinali maschili di mammiferi mediante dissociazione tissutale meccanica e citometria del flusso

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.

L'analisi di co-coltura di anello aortico: Un comodo strumento per valutare il potenziale angiogenico delle cellule stromali mesenchimali In Vitro

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.

Un modello In Vitro di un sistema di perfusione di piatti paralleli per studiare l'aderenza batterica per l'innesto di tessuti

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.

Sistema di analisi dell'angiogenesi tridimensionale utilizzando Gli spheroidi co-culturali formati da cellule formanti colonia endoteliale e cellule staminali mesenchiche

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.

Un metodo di co-cultura per studiare l'escrescenza neurite in risposta ai segnali paracrini dentali

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.

Un sistema O9-1/Hydrogel ottimizzato per lo studio dei segnali meccanici nelle cellule della cresta neurale

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.

Espressione GFP indotta dalla luce in embrioni di zebrafish utilizzando il sistema optogenetico TAEL/C120

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