Nous remercions K. et S. Je Deisseroth pour les constructions lentiviraux ChR2 et Synapsin1-tdTomato respectivement, C. Chai pour le partage de l&#39;expertise et du protocole sur la génération iPSC, WY Leong pour le support technique, les membres du laboratoire Goh pour partager des réactifs et de l&#39;expertise. Ce travail a été soutenu par Abbott Nutrition et le Centre universitaire d&#39;excellence (ACE) de bourse de recherche de GlaxoSmithKline (GSK) pour ELG, par la National Research Foundation de Singapour en vertu de son programme de recherche concurrentiel (NRF 2008 NRF-CRP 002-082) pour ELG et GJA, et par le (WCI) Programme Institut de classe mondiale de la National Research Foundation de Corée (NRF) financé par le ministère de l&#39;éducation, de la science et de la technologie de Corée (MEST) (NRF Nombre Grant: WCI 2009-003) pour GJA

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No conflicts of interest declared.

Optogenetics fournit la précision temporelle et spatiale pour l&#39;activation de populations définies de neurones 13. Dans nos expériences, la totalité du champ de l&#39;objectif du microscope a été illuminée pendant 30 sec à seulement neurones corticaux photo-stimuler exprimant CHR2. Cela nous a permis de déterminer si les connexions synaptiques ont été formés entre les différentes populations de neurones dans le co-culture. Nos résultats ont révélé que la photostimulation augmente la fréquence de la CFP dans les neurones iPSC dérivés, ce qui démontre que ces neurones reçoivent entrée synaptique de neurones corticaux présynaptiques exprimant ChR2. Ceci, à son tour, a établi que les neurones iPSC dérivés intégrés avec succès dans des circuits avec les neurones corticaux. Il ya plusieurs étapes critiques pour la génération de ce système de co-culture. Il est important de se assurer que les cultures d&#39;astrocytes souris NPC dérivées sont en bonne santé, à la mort cellulaire minimal, avant de procéder à placaged&#39;autres types de cellules. Les neurones corticaux et PNJ humains attachent bien sur astrocytes sains et enregistrements électrophysiologiques dépendent fortement des conditions de culture. Les autres étapes clés de ce protocole sont les électroporation et le placage des neurones corticaux sur les cultures d&#39;astrocytes. Comme un grand nombre de cellules meurent après électroporation, il est important de se assurer qu&#39;au moins 6.000.000 neurones corticaux sont électroporation pour le placage sur des cultures d&#39;astrocytes dans des plaques à 24 puits. Nous avons observé que lorsque moins de 6 millions de cellules ont été soumises à une électroporation, le nombre de neurones qui survivent ne forme pas un réseau neuronal dense, qui a touché des enregistrements électrophysiologiques. Le nombre de neurones corticaux électroporation devrait aussi être compatible pour chaque expérience de co-culture à permettre une comparaison entre les différentes études. Pour toutes les mesures impliquant la digestion enzymatique, il est essentiel de ne pas laisser des cellules en solution digestif pendant trop longtemps car il peut conduire à la mort cellulaire. Cetteapproche peut être utilisée pour cribler la capacité des neurones à partir de fibroblastes dérivés spécifiques au patient pour former des contacts synaptiques avec d&#39;autres neurones. Les neurones issus de fibroblastes de patients atteints de troubles neurologiques du développement du syndrome de Rett (RTT) présentent des défauts de transmission synaptique: RTT neurones présentent une diminution significative de la fréquence et de l&#39;amplitude de la CFP par rapport aux neurones sains, probablement en raison de la connectivité synaptique moins 14. Notre système de co-culture permet l&#39;examen des effets des médicaments sur les neurones présentant des défauts de développement. Ceci peut être réalisé par addition de composés d&#39;intérêt au cours de l&#39;ensemencement de NPC humains malades ainsi que pendant une exposition continue à des composés pendant toute la durée de la différenciation neuronale. Bien que ce système de co-culture peut également être utilisé comme modèle pour le dépistage de drogues, une limitation de cette approche est que le processus d&#39;étudier les effets de chaque composé candidat peut être long et fastidieux quece ne est pas une méthode de criblage à haut débit. Après le traitement avec des composés candidats, les neurones iPSC dérivés doivent être patché individuellement afin de déterminer les effets de chaque composé sur les CSP. En outre, il ne existe actuellement pas beaucoup de fibroblastes disponibles et CSPi de patients. Par conséquent, il sera d&#39;un intérêt dans un avenir proche pour avoir plus de cellules de patients et aussi pour adapter ce protocole pour le criblage à haut débit. Par exemple, par marquage de neurones COPSi dérivés avec un indicateur d&#39;activité, tels que des capteurs génétiquement codés d&#39;ions ou de potentiel de membrane, il serait possible d&#39;augmenter le débit sensiblement côte-pas à pas de la procédure d&#39;électrophysiologie de temps. Comme l&#39;ensemble du processus de génération de ce système de co-culture, de la reprogrammation des fibroblastes d&#39;effectuer des enregistrements électrophysiologiques, nécessite plus de 90 jours, il est recommandé que soit les CSPi humaines ou PNJ être élargis, après la reprogrammation et les étapes de l&#39;induction neurale, respectivement,cryoconservés et de réduire le temps nécessaire pour les expériences futures. Dans nos expériences, nous avons exprimé dans les neurones corticaux ChR2 sous le promoteur de synapsin1. Parce que ce promoteur est pan-neuronal, nous étions sans doute photostimuler deux neurones corticaux excitateurs et inhibiteurs. Si le but est de futures expériences d&#39;interroger spécifiquement circuits excitateurs ou inhibiteurs soit, les promoteurs plus spécifiques peuvent être utilisés. Alternativement, les neurones corticaux peuvent être obtenus à partir transgénique (ou virus injecté les souris) où l&#39;expression ChR2 se adresse particulièrement aux sous-populations génétiquement définies de neurones. Par exemple, les tissus corticaux récolte d&#39;une souris qui a Cre recombinase exprimés en interneurones contenant parvalbumin-(interneurones PV) qui est croisé avec une souris avec floxé ChR2 donnera une situation où les seuls neurones corticaux exprimant ChR2 seront interneurones PV 15. L&#39;utilisation de ces interneurones exprimant CHR2 dans notre co-cusystème de lture permettra de déterminer si un neurone iPSC dérivés patchée est capable d&#39;intégrer dans un circuit avec interneurones PV. Basé sur une étude précédente 9, les neurones corticaux sont matures avec chevauchement dendrites après 14 jours in vitro. Ainsi, si photostimulation ne suscite pas une réponse d&#39;un neurone iPSC dérivés patchée, il doit indiquer que le neurone n&#39;a pas la capacité de faire des connexions synaptiques avec les neurones corticaux. Un neurone iPSC dérivés est capable de recevoir une entrée synaptique soit d&#39;autres neurones iPSC dérivés ou neurones corticaux. Si les enregistrements de patch clamp classique ont été utilisés, il ne serait pas possible de distinguer l&#39;entrée synaptique où vient. L&#39;avantage de notre système est que les réponses post-synaptiques depuis l&#39;entrée présynaptique corticale peuvent être évoqués de manière sélective et identifiés. Les procédures décrites ici fournissent une approche simple pour évaluer la capacité des neurones iPSC dérivés d&#39;intégrerdans un réseau neuronal définie.

Au cours des dernières années, notre compréhension des neurones et la fonction de circuit neuronal ont été révolutionné par plusieurs outils optogénétiques qui contrôlent l&#39;excitabilité neuronale. Un tel outil est le canal de cations activé par la lumière, channelrhodopsin-2 (ChR2): neurones exprimant CHR2 potentiels d&#39;action de feu en réponse à une stimulation lumineuse 1-3. Parce que les neurones ne sont pas habituellement sensible à la lumière, cette capacité remarquable ouvre de nouvelles voies pour le contrôle précis temporelle et spatiale de l&#39;activité de populations génétiquement définies de neurones 4 et a des études de la fonction cérébrale grandement accéléré. ChR2 a été appliqué à les cellules souches à des fins différentes, de surveiller le développement neuronal 5 à réglementer l&#39;activité des réseaux de neurones 6. Ici, nous avons utilisé CHR2 pour augmenter l&#39;utilité d&#39;un système de co-culture neuronale. Systèmes de co-culture permettent l&#39;évaluation des interactions entre les différents types de cellules.Co-cultures de neurones et cellules gliales, les principaux types de système nerveux cellulaires, sont couramment utilisées pour étudier la signalisation entre ces différents types de cellules, ainsi que d&#39;évaluer l&#39;importance de cette signalisation pour les réactions physiologiques, la propagation des dommages et d&#39;examiner la mécanismes moléculaires qui sont potentiellement impliqués dans cette signalisation 7. Une étude antérieure a montré que iPSCs humains se différencient en neurones très rapidement en présence de culture contenant les neurones corticaux primaires de rat, les astrocytes, les oligodendrocytes, la microglie et 8. Dans ce protocole, nous démontrons une méthode qui utilise CHR2 dans un système de co-culture pour interroger des connexions synaptiques entre les neurones corticaux de rat et les neurones dérivés de cellules souches pluripotentes humaines induites (CISP). Nous décrivons les étapes pour disséquer et les tissus du cerveau de récolte 9 ainsi que de maintenir et de différencier les cellules progénitrices neurales de souris (PNJ) dans les astrocytes et les PNJ humains Into neurones pour créer le système de co-culture. Pour établir ce système de co-culture, nous avons utilisé la méthode de reprogrammation sans intégration décrit par Okita et al. 10 à générer des CSPi humaines. Ces CSPi ont ensuite été efficacement différenciées en PNJ dans des conditions définies chimiquement en utilisant des inhibiteurs de petites molécules comme décrit par Li et al. 11 Dans les co-cultures, les différentes populations de neurones peuvent être identifiés par l&#39;intermédiaire de détection de l&#39;expression ChR2 dans les neurones corticaux et le marquage des neurones iPSC dérivés via la protéine fluorescente, tandem dimère tomate (tdTomato). Combinant des enregistrements électrophysiologiques des neurones iPSC dérivés avec photostimulation optogenetic des neurones corticaux évaluation de la capacité de ces deux types de neurones pour former des circuits avec l&#39;autre permet. Plus précisément, photostimulation de ChR2 exprimant neurones corticaux évoquer les réponses synaptiques dans les neurones iPSC dérivés qui se sont formés des circuits synaptiquesavec le réseau de neurones corticaux photostimulée. Nous démontrons que l&#39;augmentation des courants postsynaptiques sont en effet détectés dans les neurones iPSC dérivés dans de telles conditions. Ce protocole permet l&#39;évaluation du circuit synaptique sous une variété de conditions expérimentales, y compris récapitulation des différents modèles de maladies neurologiques en utilisant CSPi dérivés à partir de fibroblastes de patients atteints de maladies.

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.

Ici, un protocole décrivant les multiples étapes de génération d&#39;une co-culture consistant en les astrocytes, des neurones corticaux humains et des neurones est illustré. CSPi humaines ont été obtenues par la reprogrammation des fibroblastes en utilisant épisomique des vecteurs 10 et précisées dans une lignée de neurones avec un cocktail d&#39;inhibiteurs de petites molécules, faisant 11 PNJ qui ont été maintenues à haute densité (figure 1A). Plusieurs étapes sont nécessaires pour générer la co-culture: la différenciation des PNJ de souris en astrocytes, le placage de neurones corticaux de rat exprimant ChR2, et l&#39;ensemencement de PNJ humains marqués avec tdTomato (figure 1B). Au jour 2 de la différenciation, la protéine acide fibrillaire gliale (GFAP) + astrocytes peuvent être facilement observés dans les cultures (figure 1C). Souris PNJ ont été autorisés à différencier pendant au moins une semaine avant l&#39;étalement des neurones corticaux exprimant ChR2 sur l&#39;astrocyte monocouche (figure 1D). Après 34 semaines de différenciation PNJ humain, les neurones tdTomato + ont été détectés dans les cultures (Figure 1E). Ce système de co-culture permet une détection cohérente des courants post-synaptiques de neurones iPSC dérivés individuels en réponse à une stimulation lumineuse (figure 2A). Quand ils sont stimulés par la lumière, des potentiels d&#39;action ont été générées dans les neurones corticaux exprimant CHR2 (figure 2B). iPSC dérivés neurones sont également excitables (figure 2C), montrant une action accrue tir potentiel que l&#39;amplitude des impulsions de courant de dépolarisation a été augmenté (figure 2D). Ainsi, ces cellules présentent des propriétés de neurones matures. En l&#39;absence de lumière, les neurones iPSC dérivés reçu entrées synaptiques spontanés (figure 2E). Ces courants intérieure post-synaptiques ont été principalement médiées par des récepteurs AMPA, parce courants à travers les récepteurs GABA serait vers l&#39;extérieur et récepteurs NMDA ne portent pas un courantt le potentiel de maintien de -70 mV. Au moins certains de ces entrées étaient de neurones corticaux présynaptiques exprimant ChR2, parce que la stimulation de lumière considérablement augmenté la fréquence des courants postsynaptiques (figure 2E). Le cours de temps de cette augmentation de l&#39;apport synaptique est illustré à la figure 2F: photostimulation des neurones corticaux a été soutenue tout au long du 30 sec à long flash de lumière. Bien que la fréquence des CSP a été élevé lors ChR2 exprimant neurones corticaux ont été photostimulée (figure 2G), leur amplitude n&#39;a pas été affectée (figure 2H). En résumé, ces résultats indiquent que les neurones COPSi dérivés présentaient des propriétés de neurones matures et pourraient former des connexions synaptiques avec les neurones corticaux présynaptiques. <img alt="Figure 1" src="/files/ftp_upload/52408/52408fig1highres.jpg" /> Figure 1: Représentation schématique pour la production de co-culturesystème. (A) Timeline pour reprogrammer des fibroblastes humains en CSPi et l&#39;induction neurale aux PNJ. (B) Échéancier de différenciation terminale des PNJ humaines en neurones matures sur neurones et astrocytes cultures corticales. (C) Souris PNJ différencier rapidement dans GFAP + astrocytes après deux jours in vitro. (D) Après une semaine, les neurones corticaux exprimant ChR2 ont été étalées sur GFAP + astrocytes. (E) À 4-5 semaines après la différenciation des PNJ humains, les enregistrements électrophysiologiques ont été réalisées sur tdTomato + neurones. Barres d&#39;échelle représentent 100 um. <img alt="Figure 2" src="/files/ftp_upload/52408/52408fig2highres.jpg" /> Figure 2: Analyse optogénétiques de la connectivité synaptique fonctionnelle. (A) iPSC-cellules dérivées marqués avec tdTomato (rouge) ont été co-cultivées avec des neurones corticaux exprimant le activé par la lumièrecanal, ChR2 (vert). Enregistrements de neurones iPSC dérivés révélé (CFP) réponses actuelles post-synaptiques, qui pourrait provenir soit les neurones corticaux ou d&#39;autres neurones iPSC dérivés. (B) Illumination (barre bleue) a évoqué une série de potentiels d&#39;action dans les neurones corticaux exprimant ChR2. ( cellules C) iPSC dérivés sont évoqués par injection de courant. (D) vs tir courbe de courant de cellules iPSC dérivés. (E) Photostimulation des neurones corticaux ChR2 exprimant (barre bleue) a augmenté la fréquence des guichets uniques dans les neurones iPSC dérivés . bien sûr (F) Temps de l&#39;augmentation de la fréquence produite par photostimulation CFP. Bleu barre indique moment de photostimulation. (G, H) Bien que la fréquence de la CFP a été augmenté de photostimulation (G), l&#39;amplitude de la CFP n&#39;a pas été affectée (H). * Indique p &lt;0,05 par rapport à contrôler avec le t-test de 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.

Une approche pour l&#39;évaluation optogénétiques formation des connexions neuronales dans un système de co-culture

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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13.3K vues.  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. 

Vidéo: 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.

Culture et co-culture de souris ovaires et follicules ovariens

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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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.

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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.

Approche précise de l&#39;ablation cellulaire pour la modélisation des lésions rénales aiguës dans le développement du poisson zèbre

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.

Une approche standardisée pour la purification multispécifique des cellules germinales masculines de mammifères par dissociation mécanique des tissus et cytométrie de flux

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.

Le dosage de co-culture anneau aortique : Un outil pratique pour évaluer le potentiel angiogénique de cellules stromales mésenchymateuses 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 modèle In Vitro d’un système de Perfusion de plaques parallèles pour étudier l’adhérence bactérienne pour greffer des tissus

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.

Système d'analyse d'angiogenèse tridimensionnel utilisant des sphéroïdes de co-culture formés par des cellules de formation de colonies endothéliales et des cellules souches mésenchymales

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.

Une méthode de co-culture pour étudier la croissance de la neurite en réponse aux signaux dentaires de paracrine de pulpe

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 système O9-1/Hydrogel optimisé pour l’étude des signaux mécaniques dans les cellules de la crête 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.

Expression de GFP induite par la lumière dans des embryons de poisson-zèbre à l’aide du système optogénétique TAEL/C120

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