Ce projet a été financé par une subvention de la Société canadienne de la sclérose d&#39;RKRWO est récipiendaire d&#39;une bourse d&#39;études de la Société canadienne de la sclérose. DTS est un récipiendaire de bourses postdoctorales de la Multiple Sclerosis Society of Canada et les Instituts de recherche en santé.

<ol>
	<li>Avellana-Adalid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+rat+oligodendrocyte+progenitors+into+proliferative+"oligospheres"+that+retain+differentiation+potential.">Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential.</a> J Neurosci Res. 45, 558-570 (1996).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85, 890-902 (1980).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+control+of+cell+survival+in+the+oligodendrocyte+lineage.">Cell death and control of cell survival in the oligodendrocyte lineage.</a> Cell. 70, 31-46 (1992).</li><li>Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43, 183-1891 (2004).</li><li>Camara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+axoglial+interactions+initiate+myelination+in+the+central+nervous+system.">Integrin-mediated axoglial interactions initiate myelination in the central nervous system.</a> J Cell Biol. 185, 699-712 (2009).</li><li>Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+promote+myelination+in+response+to+electrical+impulses.">Astrocytes promote myelination in response to electrical impulses.</a> Neuron. 49, 823-832 (2006).</li><li>Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat Protoc. 2, 1044-1051 (2007).</li><li>Pedraza, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+characterization,+and+efficient+transfection+of+highly+pure+oligodendrocyte+precursor+cultures+from+mouse+embryonic+neural+progenitors.">Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors.</a> Glia. 56, 1339-1352 (2008).</li><li>Lewin, G. R., Ritter, A. M., Mendell, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+nerve+growth+factor+in+the+development+of+myelinated+nociceptors.">On the role of nerve growth factor in the development of myelinated nociceptors.</a> J Neurosci. 12, 1896-1905 (1992).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vitro+studies+on+the+nerve+growth+factor+(NGF)+requirement+of+neurons.+II.+Sensory+neurons.">Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons.</a> Dev Biol. 58, 106-113 (1977).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Ce rapport décrit une méthode pour isoler les OPC murin de différenciation dans les cultures OL-enrichi ou OL / DRGN co-cultures. Lorsqu&#39;elle est cultivée seule, l&#39;OPC se différencier en MBP + LO VE, produisant de la myéline comme des feuilles membraneuses. Lorsqu&#39;il est ajouté à DRGN lits neurites, LO enveloppent les neurites DRGN avec MBP + membrane ve. Ce modèle profite à l&#39;enquête sur les fondements complexes qui régissent l&#39;OL à médiation ensheathment axonale. Tout d&#39;une grande valeur, l&#39;établissement de ces cultures est techniquement difficile. En particulier, les aspects exigeants incluent la digestion des tissus efficace / dissociation, l&#39;entretien de la culture médiatique au pH équilibré, et DRGN changements médias. Il est important de considérer que la durée de la digestion, la quantité de tissu étant digérée et le montant de la trituration affecte le résultat d&#39;efficacité et à la fin de la dissociation des tissus. Il n&#39;est pas inhabituel pour les chercheurs expérimentés d&#39;obtenir de faibles rendements cellulaires des tissus du système nerveux dissociés. En outre, les OPC murins ont tendance à être sensibles aux changements dans le pH du milieu de culture, en particulier dans des conditions alcalines. L&#39;entretien des cultures, à 8,5% de CO 2 vise à empêcher cela, puisque les OPC semblent mieux tolérer des conditions légèrement acides au cours de base. En ce qui concerne DRGNs alimentation, les changements de supports doivent être effectués rapidement pour ne pas dessécher les neurones, cependant, doit être doux pour ne pas perturber le lit de neurites en développement. Changements brusques des médias peuvent déloger le lit neurites du substrat, et le résultat probable dans sa dissociation complète de la lamelle. Le mérite potentiel de ce système modèle éclipse fortement son caractère techniquement exigeante. Un avantage de ce système est l&#39;utilisation de la post-natale des souris pour la dérivation de la culture cellulaire, en contournant la nécessité de sacrifier les femelles reproductrices de récolter des tissus embryonnaires. Un autre avantage est l&#39;absence d&#39;obligation pour les facteurs de croissance (GFS) pour l&#39;expansion de l&#39;OPC. Mixte cultures gliales fournir un environnement qui favorise la propagation des OPC, vraisemblablement en raison de la présence de facteurs trophiques astrocyte dérivées. D&#39;autres méthodes, telles que la dérivation via neurosphères 7,8, reposent sur ​​les propriétés mitogéniques de GFS tels que le facteur de croissance fibroblastique basique (bFGF), facteur de croissance épidermique (EGF) et le facteur de croissance dérivé des plaquettes (PDGF) pour l&#39;expansion du CPVP. De même, en utilisant le développement postnatal (P5-10) pour les souris DRGNs évite l&#39;obligation de compléter les milieux de culture avec un facteur de croissance nerveuse (NGF), un facteur neurotrophique requis pour le taux de survie in vitro des embryons DRGNs 9, 10. Il est intéressant d&#39;éviter d&#39;utiliser le NGF car elle influence négativement la capacité de myélinisation des LO lorsqu&#39;elles sont cultivées avec des DRGNs 4. Éviter l&#39;utilisation du GF-complété des médias a aussi des avantages économiques, comme ces réactifs devenir coûteux s&#39;il est utilisé à grande échelle. Peut-être l&#39;avantage le plus important de ce modèle est sa culture de dérivation de la souris uniquement les tissus, offrant ainsi la possibilité de tirer à la fois les OPC et les DRGNs de la grande variété de lignées de souris transgéniques. Cela permet pour l&#39;étude de deux DRGN et / ou OPC propriétés spécifiques qui régissent la myélinisation. Cela sera particulièrement important pour l&#39;élucidation des interactions récepteur / ligand régulation OL-médiée myélinisation des axones. En tout, cette technique est d&#39;une grande valeur en ce qui concerne la recherche en neurosciences en raison de ses applications vers la compréhension des signaux moléculaires qui sous-tendent la myélinisation.

<html><table border="1"><tbody><tr><td> Nom du produit </td><td> Société </td><td> Numéro de produit </td></tr><tr><td> Eagle modifié par Dulbecco (DMEM) </td><td> Multicell </td><td> 319 à 005-CL </td></tr><tr><td> Hank Balanced Salt Solution (HBSS) </td><td> Invitrogen </td><td> 14170-112 </td></tr><tr><td> Minimum Essential Media (MEM) </td><td> GIBCO </td><td> 12360-038 </td></tr><tr><td> Sérum fœtal bovin (FBS) </td><td> GIBCO </td><td> 10091-148 </td></tr><tr><td> Pénicilline-streptomycine (Pen / Strep) </td><td> GIBCO </td><td> 15140-122 </td></tr><tr><td> Glutamax </td><td> Invitrogen </td><td> 35050-061 </td></tr><tr><td> Poly-L-lysine </td><td> Sigma-Aldrich </td><td> P2636 </td></tr><tr><td> Albumine sérique bovine (BSA) </td><td> Sigma-Aldrich </td><td> A4503 </td></tr><tr><td> Homme mérosine protéine purifiée (LN2) </td><td> Millipore </td><td> CC085 </td></tr><tr><td> Recombinante du facteur neurotrophique rat ciliaire (CNTF) </td><td> PeproTech </td><td> 450-50 </td></tr><tr><td> L-thyroxine </td><td> Biochemika </td><td> 89430 </td></tr><tr><td> Holo-transferrine </td><td> Sigma-Aldrich </td><td> T0665 </td></tr><tr><td> B27 complément </td><td> GIBCO </td><td> 0080085-SA </td></tr><tr><td> L&#39;insuline bovine </td><td> Sigma-Aldrich </td><td> I6634 </td></tr><tr><td> 3,3 &#39;,5-triiodo-L-thyronine </td><td> Sigma-Aldrich </td><td> I6634 </td></tr><tr><td> La progestérone </td><td> Sigma-Aldrich </td><td> P8783 </td></tr><tr><td> Putrescine </td><td> Sigma-Aldrich </td><td> P7505 </td></tr><tr><td> Sélénite de sodium </td><td> Sigma-Aldrich </td><td> S5261 </td></tr><tr><td> 5-fluoro-2&#39;-désoxyuridine (FUDR) </td><td> Sigma-Aldrich </td><td> F0503 </td></tr><tr><td> Solution de papaïne </td><td> Worthington </td><td> LS003126 </td></tr><tr><td> DNaseI </td><td> ROCHE </td><td> 1010159001 </td></tr><tr><td> L-cystéine </td><td> Sigma-Aldrich </td><td> C7352 </td></tr><tr><td> 24 puits de culture tissulaire </td><td> Cellstar </td><td> 662-160 </td></tr><tr><td> Flacons de culture de tissu T25 avec capuchon de l&#39;évent </td><td> Corning </td><td> 430639 </td></tr><tr><td> Boîtes de 10 cm de culture de tissu </td><td> Corning </td><td> 430167 </td></tr><tr><td> 10 cm de Petri </td><td> Fisher Scientific </td><td> 0875713 </td></tr><tr><td> Une collagénase </td><td> ROCHE </td><td> 103578 </td></tr><tr><td> CellTrics 50 um (optionnel) de filtre </td><td> PARTEC </td><td> 04-004-2327 </td></tr><tr><td> La myéline protéine basique (MBP) Anticorps </td><td> AbD Serotec </td><td> MCA409S </td></tr><tr><td> NG2 d&#39;anticorps </td><td> Millipore </td><td> AB5320 </td></tr><tr><td> La myéline-associated glycoprotein (MAG) antibody </td><td> Millipore </td><td> MAB1567 </td></tr><tr><td> 2 &#39;, 3&#39;-cyclique nucléotide 3&#39;-phosphodiestérase (CNP) antibody </td><td> Covance </td><td> SMI-91R-100 </td></tr><tr><td> L&#39;actine pan Ab-5 anticorps </td><td> Fitzgerald </td><td> 10R-A106AX </td></tr><tr><td> Neurofilament-200 (NF) antibody </td><td> Sigma-Aldrich </td><td> N4142 </td></tr><tr><td> Bêta-tubuline trois chaînes (Tuj1) antibody </td><td> Millipore </td><td> MAB5544 </td></tr><tr><td> Alexa Fluor 488 chèvre anti-lapin IgG (H + L) anticorps secondaire </td><td> Invitrogen </td><td> A11008 </td></tr><tr><td> Alexa Fluor 555 chèvre anti-souris IgG (H + L) anticorps secondaire </td><td> Invitrogen </td><td> A21422 </td></tr><tr><td> Alexa Fluor 647 chèvre anti-rat (IgG) (H + L) anticorps secondaire </td><td> Invitrogen </td><td> A21247 </td></tr><tr><td> Chèvre anti-souris IgG (H + L)-HRP anticorps secondaire conjugué </td><td> BioRad </td><td> 170-6516 </td></tr><tr><td> Chèvre anti-rat IgG (H + L)-HRP anticorps secondaire conjugué </td><td> Santa Cruz Biotechnology </td><td> SC-2065 </td></tr><tr><td> 4 &#39;,6-diamidino-2-phénylindole (DAPI) </td><td> Sigma-Aldrich </td><td> D9542 </td></tr></tbody></table> Recettes des médias OL-100X * Supplément <table border="1"><tbody><tr><td> Ingrédient </td><td> Montant à ajouter </td></tr><tr><td> DMEM </td><td> 100 ml </td></tr><tr><td> BSA </td><td> 1,02 g </td></tr><tr><td> La progestérone </td><td> 0,6 mg </td></tr><tr><td> Putrescine </td><td> 161 mg </td></tr><tr><td> Sélénite de sodium </td><td> 0,05 mg </td></tr><tr><td> 3,3 &#39;,5-triiodo-L-thyronine </td><td> 4 mg </td></tr></tbody></table> * Store à -80 ° C dans 250 aliquotes t &quot;&gt; Médias OL <table border="1"><tbody><tr><td> Ingrédient </td><td> Montant à ajouter </td></tr><tr><td> DMEM </td><td> 23,75 mL </td></tr><tr><td> OL-100X Supplément </td><td> 250 uL </td></tr><tr><td> L&#39;insuline bovine (de 1 mg / mL de stock) </td><td> 125 ul </td></tr><tr><td> Glutamax </td><td> 250 uL </td></tr><tr><td> Holo-transferrine (de 33 mg / mL de stock) </td><td> 37,5 uL </td></tr><tr><td> B27 Supplément </td><td> 500 ul </td></tr><tr><td> FBS </td><td> 125 ul </td></tr><tr><td> CNTF (de 50 ng / uL stock) </td><td> 25 uL </td></tr></tbody></table> Mixte des milieux de culture gliales (composé de DMEM) <table border="1"><tbody><tr><td> Ingrédient </td><td> La concentration finale </td></tr><tr><td> FBS </td><td> 10% </td></tr><tr><td> Pen / Strep (0,33% du stock) </td><td> 33 unités / ml de pénicilline et 33 pg / ml de streptomycine </td></tr><tr><td> Glutamax </td><td> 1% </td></tr></tbody></table> DRGN médias (constitué de DMEM) <table border="1"><tbody><tr><td> Ingrédient </td><td> La concentration finale </td></tr><tr><td> FBS </td><td> 10% </td></tr><tr><td> Pen / Strep (1% du stock) </td><td> 100 unités / ml de pénicilline et 100 pg / ml de streptomycine </td></tr></tbody></table> Recettes solution de digestion: OPC papaïne solution (composé de MEM) <table border="1"><tbody><tr><td> Ingrédient </td><td> La concentration finale </td></tr><tr><td> Solution de papaïne </td><td> 1,54 mg / mL </td></tr><tr><td> L-cystéine </td><td> 360 pg / ml </td></tr><tr><td> DNaseI </td><td> 60 pg / mL </td></tr></tbody></table> DRG papaïne solution (composé de HBSS) <table border="1"><tbody><tr><td> Ingrédient </td><td> La concentration finale </td></tr><tr><td> La papaïne </td><td> 1,54 mg / mL </td></tr><tr><td> L-cystéine </td><td> 360 pg / ml </td></tr></tbody></table> DRG collagénase Une solution (composé de HBSS) <table border="1"><tbody><tr><td> Ingrédient </td><td> La concentration finale </td></tr><tr><td> Une collagénase </td><td> 4 mg / ml </td></tr></tbody></table>

derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues

Identifier les mécanismes moléculaires qui sous-tendent le développement OL n&#39;est pas seulement essentielle pour approfondir notre connaissance de la biologie OL, mais a également des implications pour la compréhension de la pathogenèse des maladies démyélinisantes comme la sclérose en plaques (SEP). Développement cellulaire est communément étudié avec primaires modèles de culture cellulaire. Culture de cellules primaires facilite l&#39;évaluation d&#39;un type cellulaire donné en fournissant un environnement contrôlé, sans les variables exogènes qui sont présentes in vivo. Bien que les cultures OL provenant de rats ont fourni une grande quantité de comprendre la biologie de LO, des efforts similaires à établir des cultures OL de souris a été rencontré d&#39;obstacles majeurs. Développer des méthodes de culture murin LO primaire est impératif afin de profiter des lignes disponibles de souris transgéniques. De multiples méthodes pour l&#39;extraction des OPC à partir de tissus de rongeurs ont été décrites, allant de la dérivation Neurosphère, la purification de l&#39;adhérence différentielle et immunopurification 1-3. Alors que de nombreuses méthodes offrent de succès, la plupart nécessitent des temps de culture extensive et / ou du matériel coûteux / réactifs. Pour contourner cela, purifiant les OPC à partir de tissus murins avec une adaptation de la méthode initialement décrite par McCarthy &amp; de Vellis 2 est préféré. Cette méthode implique physiquement OPC séparant d&#39;une culture mixte gliales provenant cortex des rongeurs nouveau-nés. Le résultat est une population purifiée OPC qui peuvent être différenciées en une culture de l&#39;OL enrichi. Cette approche est attrayante en raison de ses temps de culture relativement courtes et l&#39;exigence inutile pour les facteurs de croissance ou des anticorps immunopanning. Tout en explorant les mécanismes du développement de LO dans une culture purifiée est informatif, il ne fournit pas l&#39;environnement le plus physiologiquement pertinents pour évaluer la formation gaine de myéline. Co-culture avec des neurones LO donnerait un aperçu des fondements moléculaires régulant l&#39;OL à médiation myélinisation des axones. Pour beaucoup de OL / neurone de co-culture des études, dorsale neurones ganglion de la racine (DRGNs) se sont révélés être le type de neurone de choix. Elles sont idéales pour la co-culture avec LO en raison de leur facilité d&#39;extraction, le montant minimal de cellules contaminantes, et la formation de neurites lits denses. Alors que les études utilisant rat / souris myélinisantes xenocultures ont été publiés 4-6, une méthode pour la dérivation d&#39;une telle OL / DRGN myélinisantes co-cultures de tissus post-natale murin n&#39;a pas été décrite. Nous présentons ici des méthodes détaillées sur la façon de produire efficacement de telles cultures, avec des exemples de résultats escomptés. Ces méthodes sont utiles pour aborder les questions pertinentes pour le développement de LO / fonction myélinisantes, et sont des outils utiles dans le domaine des neurosciences.

Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Cet article décrit les méthodes pour estimer la population enrichie des cellules précurseurs des oligodendrocytes murins (OPC) en culture primaire, qui se différencier pour produire des oligodendrocytes matures (MCO). En outre, ce rapport décrit des techniques pour produire murin myélinisantes co-cultures par semis OPC de la souris sur un lit de neurites de souris dorsale neurones ganglion de la racine (DRGNs).

Dérivation des cultures enrichies en oligodendrocytes et Oligodendrocyte / Neuron myélinisantes co-cultures de post-natal tissus murins

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

36.1K vues.  Ottawa Hospital Research Institute. Cet article décrit les méthodes pour estimer la population enrichie des cellules précurseurs des oligodendrocytes murins (OPC) en culture primaire, qui se différencier pour produire des oligodendrocytes matures (MCO). En outre, ce rapport décrit des techniques pour produire murin myélinisantes co-cultures par semis OPC de la souris sur un lit de neurites de souris dorsale neurones ganglion de la racine (DRGNs).

Vidéo: Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

La cryoconservation de tissu cortical Blocs pour la génération des cultures de neurones hautement enrichi

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Recherche

Neurosciences

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

Dérivation de précurseurs gliales restreint de souris E13

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Progénitrices dérivées de Système de culture Oligodendrocyte du cerveau foetal humain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Simultanée pré-et post-synaptique enregistrement électrophysiologique de<em&gt; Xenopus</em&gt; Nerf-muscle co-cultures

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Préparation de neurones co-cultures avec l&#39;unité portable de précision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Cultures organotypique de tranche d&#39;étudier Oligodendrocyte Dynamics et myélinisation

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Enquêter Régénération fonctionnelle en organotypiques médullaires Co-cultures Cultivé sur des tableaux multi-électrodes

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Dérivation des cultures leptoméninge explants de Postmortem humaines donateurs cerveau

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

Neurone-Macrophage co-cultures d’activer les Macrophages qui sécrètent des facteurs moléculaires avec l’activité des neurites

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

Préparation et Immunostaining des cérébelleux Myélinisantes Organotypiques

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...