Notre travail est soutenu par le Fonds national suisse (FNS), l&#39;initiative suisse en biologie des systèmes (SystemsX.ch), la Muscular Dystrophy Association aux Etats-Unis (MDA), l&#39;Association française Contre les Myopathies (AFM), le Royaume-mitochondrial Disease Foundation (UMDF), la Fondation Gebert Rüf-Programme des maladies rares (GRF), la Société suisse pour la recherche des maladies musculaires (SSEM / FSRMM), le Swiss Life &quot;Jubiläumsstiftung für Forschung Volksgesundheit und medizinische&quot;, la Roche Research Foundation et l&#39;Université de Bâle.

1. Préparation de la culture primaire de cellules musculaires humaines <ol><li> Mettre en place des cultures de cellules musculaires humaines, selon la technique explantation-re-explantation 4. Tout d&#39;abord, éliminer les obstacles non-tissu musculaire à partir des biopsies. Puis, incorporer 1 mm explants musculaires 3 dans un caillot de plasma, ce qui permet de sortir de fibroblastes des explants. </li><li> Les explants incorporés sont transférées sur un plat de plasma gélatine revêtue et laisser croître dans un milieu de croissance normale (MEM supplémenté avec 25% support 199, 10% de sérum bovin foetal [FBS], 1% de pénicilline / streptomycine, 10 pg / ml d&#39;insuline, 10 ng / ml de facteur de croissance épidermique humain [EGF], 12,5 ng / ml de facteur humain de croissance des fibroblastes [FGF]) dans une monocouche. Les cellules qui émergent à partir des explants dans ces conditions sont des cellules musculaires. Ces cellules vont continuer à croître dans une monocouche après le retrait des explants. </li><li> Culture des cellules aneurally sur 0,1% de gélatine recouvertes de plaques en milieu de croissance normal. Les cellules peuvent êtreaneurally cultivées et amplifiées selon les besoins. Il est préférable d&#39;utiliser des cellules musculaires à un passage entre 3 et 8 pour effectuer les co-cultures. Il est essentiel pour innerver myotubes jeunes, après ce point, les co-cultures ont un risque élevé d&#39;échec. En outre, les myotubes ne devrait pas être trop grand et trop épais. </li><li> Deux jours avant l&#39;innervation est prévu, plaque les cellules musculaires sur 35 mm boîtes de culture tissulaire (ou plaques à 6 puits) à une densité de 500.000 cellules par boîte de milieu de croissance normal. A cette densité, les cellules sont à confluence du bon de commencer la fusion, le lendemain, et l&#39;innervation ultérieure le jour d&#39;après. En utilisant ces conditions, les myotubes sont à la bonne étape pour l&#39;innervation optimale et donc la contraction des fibres ultérieure. </li><li> Le jour suivant, remplacer le milieu de croissance normale avec un milieu de fusion composée d&#39;MEM supplémenté avec 25% support 199, 5% de FBS, 10 pg / ml d&#39;insuline et de 1% de pénicilline / streptomycine. </li></ol> 2. Isolati<html> sur des explants embryonnaires de rat de la moelle épinière Toute la procédure d&#39;isoler les explants de moelle épinière est effectuée sous une loupe binoculaire, dans une solution saline équilibrée de Hank (HBSS, Gibco / Invitrogen ref 14170) contenant 10% de sérum de veau fœtal (FBS). <ol><li> Il est crucial que les embryons de rat sont entre ED 13 et 14, un stade de développement différent mettrait en péril l&#39;ensemble de la procédure. Sacrifiez une femme enceinte avec du CO 2 et de recueillir la chaîne embryon entier dans un milieu HBSS. Isoler chaque embryon et les décapiter pour l&#39;euthanasie. </li><li> Disséquer le cordon médullaire embryon à partir du reste du corps d&#39;une seule pièce et enlever de muscle entourant et du tissu conjonctif. Soyez très prudent de garder les ganglions de racine dorsale (DRG) rattachés à la moelle épinière afin d&#39;assurer l&#39;innervation fonctionnelle. </li><li> Couper chaque moelle épinière transversalement de manière que chaque explant contient au moins deux DRG attaché (explants de 1 mm 3). </li></ol><html> ove_title &quot;&gt; 3. Innervation des cellules musculaires humaines avec des explants embryonnaires de rat de la moelle épinière <ol><li> Retirer le milieu de fusion de la culture des cellules musculaires, en laissant une fine couche de support sur la monocouche. </li><li> Placez les explants attentivement sur la couche de cellules musculaires, cinq explants régulièrement espacés par boîte de 35 mm. </li><li> Ajouter très lentement, goutte à goutte milieu de fusion, sur chaque explant. Il est nécessaire d&#39;ajouter moyenne pour les cellules musculaires pour survivre, mais pas trop afin de permettre aux explants d&#39;adhérer d&#39;abord et prendre contact avec les cellules. Si le milieu n&#39;est pas ajouté correctement (trop fort ou trop moyennes), les explants va flotter et ne parviendra pas à innerver les cellules musculaires. </li><li> Une fois que les explants de commencer à respecter, le haut le milieu de fusion à 2 ml par boîte de 35 mm, et mis les petits plats très soigneusement de nouveau dans l&#39;incubateur. </li></ol> 4. Entretien des hétérologues nerf-muscle co-cultures <ol><li> Changer le milieu de fusion deux fois par semaine. </li></ol>_title &quot;&gt; Résultats représentatifs 5. Dès que deux jours après l&#39;innervation, il est possible de voir les neurites émanant de chaque explant et établir des contacts avec les cellules musculaires, représentés par les structures sur le bouton-like (figure 1). Dès 4-6 jours après l&#39;innervation, quelques fibres individuelles va commencer à se contracter. Les contractions persistent pendant plusieurs mois, maintenu en changeant simplement le milieu de culture deux fois par semaine. Après environ 2 semaines, l&#39;explant la moelle épinière se dégénère, mais l&#39;innervation est conservée. Ceci est normal. Une fine couche de composant nerf reste à la surface de la couche de cellules musculaires. Ce réseau nerveux est suffisante pour maintenir la fonctionnalité de l&#39;unité motrice. Si les explants flottent dans le milieu de la journée après l&#39;innervation, ils ne seront jamais respecter. Cela se produit lorsque la couche de cellules musculaires estpas suffisamment confluentes, ou lorsque le milieu est ajouté trop rudement. Parfois, les explants adhère, mais pas de neurites vont émerger. Cela est probablement dû à l&#39;absence de GHM qui s&#39;y rattachent. Dans ce cas aussi, l&#39;innervation ne sera pas établie. Manipuler les plats très attentivement la première semaine, comme les explants peuvent encore se détachent facilement. Pour échanger les médias de fusion, ajoutez le liquide goutte à goutte avec une vitesse minimale pour éviter le détachement des cellules. <img alt="Figure 1" src="/files/ftp_upload/3616/3616fig1.jpg" /> Figure 1. Moelle épinière explants-cellules musculaires des co-cultures à partir du jour 1 au jour 15.

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Un outil physiologique in vitro pour étudier la fonction des cellules musculaires dans le contexte normal et pathologique est du plus haut intérêt pour les myologues, parce que les cultures de cellules musculaires ne sont généralement pas récapituler l&#39;importance de plusieurs cellules et des cellules de type de connexions. L&#39;ajout de purifier les neurones moteurs dans les cellules musculaires ne suffit pas pour parvenir à une unité motrice fonctionnelle puisque la présence de cellules ...

<table border="1"><tbody><tr><td> Nom de réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> HBSS </td><td> Gibco / Invitrogen </td><td> 14170 </td></tr><tr><td> MEM </td><td> Gibco / Invitrogen </td><td> 31095 </td></tr><tr><td> Milieu 199 </td><td> Gibco / Invitrogen </td><td> 31153 </td></tr><tr><td> Sérum fœtal bovin </td><td> Clone fœtale Perbio </td><td> SH30066.03 </td></tr><tr><td> Insuline </td><td> Sigma </td><td> I9278 </td></tr><tr><td> EGF humain </td><td> Sigma </td><td> E9644 </td></tr><tr><td> Human FGF </td><td> Sigma </td><td> F0291 </td></tr><tr><td> Pénicilline / streptomycine solution </td><td> Gibco </td><td> 15140 </td></tr></tbody></table>

a functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells

Homme cellules musculaires primaires en culture en monocouche aneurally rarement contrat spontanément en raison, en l&#39;absence d&#39;une composante du nerf, la différenciation cellulaire est limitée et la stimulation des neurones moteurs est une manquante. Ces limitations entravent l&#39;étude in vitro de nombreuses maladies neuromusculaires dans les cellules musculaires en culture. Surtout, les contraintes expérimentales de monocouches, les cellules musculaires en culture peuvent être surmontés par l&#39;innervation fonctionnelle des fibres musculaires avec des explants de moelle épinière dans les co-cultures. Ici, nous montrons les différentes étapes nécessaires à la réalisation efficace, innervation correcte de l&#39;homme des cellules musculaires primaires, menant à une contraction de la différenciation et la fibre selon la méthode développée par Askanas 2. Pour ce faire, les cellules musculaires sont co-cultivées avec des explants de moelle épinière d&#39;embryons de rat chez ED 13,5, avec les ganglions de la racine dorsale encore attaché aux tranches de la moelle épinière. Après quelques jours, le muscle fibres de commencer à contrat et devenir contre-striée par innervation par neurites fonctionnels saillie à partir des explants de moelle épinière qui se connectent à des cellules musculaires. Cette structure peut être maintenue pendant de nombreux mois, tout simplement par un échange régulier du milieu de culture. Les applications de cet outil précieux sont nombreux, car il représente un modèle fonctionnel pour des analyses multidisciplinaires de développement musculaire humaine et l&#39;innervation. En fait, un complet de novo d&#39;installation de la jonction neuromusculaire se produit dans une boîte de culture, ce qui permet une mesure facile de nombreux paramètres à chaque étape, dans un contexte fondamental et physiologique. Pour ne citer que quelques exemples, génomique et / ou des études protéomiques peuvent être effectuées directement sur les co-cultures. En outre, les effets pré-et post-synaptique peut être évalué précisément et séparément à la jonction neuromusculaire, parce que les deux composants proviennent d&#39;espèces différentes,rat et l&#39;homme, respectivement. Le nerf-muscle de co-culture peut également être effectuée avec des cellules musculaires humaines isolées chez des patients souffrant de maladies neuromusculaires du muscle ou 3, et peut donc être utilisé comme un outil de dépistage pour les médicaments candidats. Enfin, aucun équipement spécial, mais un habitué BSL2 installation est nécessaire pour reproduire une unité motrice fonctionnelle dans une boîte de culture. Cette méthode est donc précieuse tant pour le muscle ainsi que les communautés de recherche neuromusculaire pour des études physiologiques et mécaniste de la fonction neuromusculaire, dans un contexte normal et la maladie.

Watch this Scientific Journal Video about A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells at JoVE.com

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Les cellules musculaires en culture sont un modèle inadéquat de récapituler les muscles innervés<em&gt; In vivo</em&gt;. Une unité fonctionnelle du moteur peut être reproduite<em&gt; In vitro</em&gt; Par l&#39;innervation de l&#39;homme les cellules musculaires différenciées primaires à l&#39;aide de rats explants d&#39;embryons de la moelle épinière. Cet article décrit comment les co-cultures d&#39;explants de moelle épinière et les cellules musculaires sont établis.

Une unité fonctionnelle du moteur dans la boîte de culture: La co-culture d&#39;explants de la moelle épinière et les cellules musculaires

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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15.2K vues.  University of Basel. Les cellules musculaires en culture sont un modèle inadéquat de récapituler les muscles innervés In vivo. Une unité fonctionnelle du moteur peut être reproduite In vitro Par l'innervation de l'homme les cellules musculaires différenciées primaires à l'aide de rats explants d'embryons de la moelle épinière. Cet article décrit comment les co-cultures d'explants de moelle épinière et les cellules musculaires son...

Vidéo: A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection et la culture de neurones de la moelle épinière commissurales embryonnaires

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Dissection et la culture de cellules de ganglions et de Chick Statoacoustic explants moelle épinière dans des gels de collagène pour des essais de neurites

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embryon de poulet moelle épinière culture Tranche Protocole

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Analyse de développement germe dentaire innervation Utilisation de périphériques Co-culture microfluidique

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

poisson zèbre<em&gt; In Situ</em&gt; Préparation à la moelle épinière électrophysiologiques Les enregistrements de la colonne vertébrale et sensorielle motoneurones

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Laminectomy et implantation de fenêtre de moelle épinière dans la souris

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identification des marqueurs de surface cellulaire des cellules neurales primaires de tige et de progéniture par l'étiquetage métabolique du sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Injection directe d’un vecteur Lentiviral met en évidence plusieurs voies motrices de la moelle épinière de Rat

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

Conversion de cellules souches pluripotentes induites par l’homme (iPSCs) en neurones moteurs spinaux et crânien fonctionnels utilisant des vecteurs PiggyBac

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Livraison intra-artérielle de cellules souches neuronales au cerveau du rat et de la souris : application à l’ischémie cérébrale

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...