Nuestro trabajo es apoyado por la Swiss National Science Foundation (SNF), la Iniciativa de Suiza en Biología de Sistemas (SystemsX.ch), la Asociación de Distrofia Muscular EE.UU. (MDA), la Asociación Francesa contra las Miopatías (AFM), la United Mitochondrial Disease Foundation (UMDF), la Fundación Gebert-Ruf Enfermedades Raras Programa (GRF), la Sociedad Suiza para la Investigación de las Enfermedades Musculares (SSEM / FSRMM), el Swiss Life &quot;Jubiläumsstiftung für Medizinische Forschung und Volksgesundheit&quot;, la Roche Fundación para la Investigación y la Universidad de Basilea.

1. Preparación de la primaria la cultura humana de la célula muscular <ol><li> Establecer los cultivos de músculo de células humanas de acuerdo con la técnica de re-explantación-4 explantación. En primer lugar, eliminar los no-tejido muscular de las biopsias. A continuación, incorporar 1 mm 3 explantes de músculo en un coágulo de plasma, lo que permite salir de los fibroblastos para los explantes. </li><li> Los explantes incrustadas se transfieren a un plato recubierto de plasma gelatina y dejarlas crecer en medio de crecimiento normal (MEM suplementado con 25% medio 199, 10% de suero fetal bovino [FBS], 1% de penicilina / estreptomicina, 10 mg / ml de insulina, 10 ng / ml de factor de crecimiento epidérmico humano [EGF], 12,5 ng / ml de factor de crecimiento de fibroblastos humanos [FGF]) en una monocapa. Las células que surgen de los explantes bajo esas condiciones son las células musculares. Estas células continuará creciendo en una monocapa después de la eliminación de los explantes. </li><li> Cultivar las células aneurally en gelatina al 0,1% recubiertos con placas en medio de crecimiento normal. Las células pueden seraneurally cultivaron y se amplifica cuando sea necesario. Es mejor utilizar células del músculo en un paso entre 3 y 8 para realizar los co-cultivos. Es de suma importancia para inervar miotubos jóvenes, después de este punto, los co-cultivos tienen un alto riesgo de fracasar. Además, miotubos no debe ser demasiado grande y demasiado grueso. </li><li> Dos días antes de la inervación está previsto, la placa de las células musculares en placas de 35 mm de cultivo de tejidos (o placas de 6 pocillos) a una densidad de 500.000 células por placa en medio de crecimiento normal. En ese densidad, las células están en la confluencia adecuado para iniciar la fusión en el día siguiente, y la inervación posterior del día después. Utilizando esas condiciones, los miotubos se encuentran en la etapa correcta de la inervación y la contracción de la fibra óptima, por tanto posterior. </li><li> Al día siguiente, reemplazar el medio de crecimiento normal con un medio de fusión compuesta de MEM suplementado con 25% medio 199, 5% de FBS, insulina 10 ug / ml y 1% de penicilina / estreptomicina. </li></ol> 2. Isolati<html> el de la rata explantes embrionarias de la médula espinal Todo el procedimiento para aislar los explantes de médula espinal se realiza bajo un microscopio binocular, en solución salina equilibrada de Hank (HBSS, Gibco / Invitrogen ref 14170) que contiene 10% suero bovino fetal (FBS). <ol><li> Es crucial que los embriones de rata se encuentran entre 13 y 14 de la disfunción eréctil como una etapa de desarrollo diferente pondría en peligro todo el procedimiento. El sacrificio de una mujer embarazada de CO 2 y recoger toda la cadena de embriones en un medio de HBSS. Aislar cada embrión y decapitar a favor de la eutanasia. </li><li> Disecar el cordón espinal de embriones desde el resto del cuerpo de una sola pieza y quitar músculo que rodea y tejido conectivo. Tenga mucho cuidado de mantener los ganglios de raíz dorsal (GRD) conectados a la médula espinal para garantizar la inervación funcional. </li><li> Corte cada médula espinal transversalmente de manera que cada explante contiene al menos 2 DRG unido a él (explantes de 1 mm 3). </li></ol><html> ove_title &quot;&gt; 3. Inervación de las células musculares embrionarias humanas con explantes médula espinal de ratas <ol><li> Eliminar el medio de fusión en el cultivo de células del músculo, dejando una fina capa de medio sobre la monocapa. </li><li> Coloque los explantes con cuidado en la capa de células musculares, cinco explantes uniformemente espaciados por placa de 35 mm. </li><li> Añadir muy lentamente, gota a medio sabio, la fusión en cada explante. Es necesario añadir medio para las células musculares para sobrevivir, pero no demasiado para permitir que los explantes de adherirse primero y hacer contacto con las células. Si el medio no se agrega correctamente (demasiado fuerte o demasiado medio), los explantes se flotará y no podrá inervan las células musculares. </li><li> Una vez que los explantes de empezar a cumplir, arriba del medio de la fusión de 2 ml por placa de 35 mm, y poner los platos con mucho cuidado de nuevo en la incubadora. </li></ol> 4. El mantenimiento de los heterólogos nervio-músculo Co-culturas <ol><li> Cambiar el medio de la fusión dos veces por semana. </li></ol>_title &quot;&gt; 5. Los resultados representativos Tan pronto como dos días después de la inervación, es posible ver las neuritas que salen de cada explante y establecer contactos con las células musculares, representadas por las estructuras como botón-(Fig. 1). Ya en 4-6 días después de la inervación, unos pocos fibras individuales comenzará a contraerse. Las contracciones persistir durante muchos meses, mantiene simplemente cambiando el medio de cultivo dos veces a la semana. Después de aproximadamente 2 semanas, el explante de la médula espinal se degenera, pero la inervación se conserva. Esto es normal. Una fina capa de componente nervio queda en la superficie de la capa de células del músculo. Esta red nervioso es suficiente para mantener la funcionalidad de la unidad de motor. Si los explantes flotar en el medio el día después de inervación, nunca se adherirá. Esto ocurre cuando la capa de células del músculo esno suficientemente confluente, o cuando el medio se añade demasiado áspero. A veces, los explantes se adhiere, pero no saldrá neuritas. Esto es probablemente debido a la falta de DRG adjunto a la misma. En este caso también, la inervación no se establecerá. Manipular los platos con mucho cuidado la primera semana, ya que los explantes todavía se puede separar fácilmente. Para intercambiar los medios de fusión, añadir el líquido gota a gota con una velocidad mínima para evitar el desprendimiento de células. <img alt="Figura 1" src="/files/ftp_upload/3616/3616fig1.jpg" /> Figura 1. De la médula espinal, las células musculares explantes co-cultivos a partir del día 1 al día 15.

<ol>
	<li>Delaporte, C., Dautreaux, B., Fardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+myotube+differentiation+in+vitro+in+different+culture+conditions.">Human myotube differentiation in vitro in different culture conditions.</a> Biol. Cell. 57, 17-22 (1986).</li><li>Kobayashi, T., Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+muscle+cultured+in+monolayer+and+cocultured+with+fetal+rat+spinal+cord:+importance+of+dorsal+root+ganglia+for+achieving+successful+functional+innervation.">Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation.</a> J. Neurosci. 7, 3131-3141 (1987).</li><li>Braun, S., Croizat, B., Lagrange, M. C., Warter, J. M., Poindron, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+muscular+abnormalities+in+culture+in+spinal+muscular+atrophy.">Constitutive muscular abnormalities in culture in spinal muscular atrophy.</a> Lancet. 345, 694-695 (1995).</li><li>Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+program+for+investigating+adult+human+skeletal+muscle+grown+aneurally+in+tissue+culture.">A new program for investigating adult human skeletal muscle grown aneurally in tissue culture.</a> Neurology. 25, 58-67 (1975).</li><li>Guettier-Sigrist, S., Coupin, G., Warter, J. M., Poindron, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+types+required+to+efficiently+innervate+human+muscle+cells+in+vitro.">Cell types required to efficiently innervate human muscle cells in vitro.</a> Exp. Cell Res. 259, 204-212 (2000).</li><li>Lefebvre, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+spinal+muscular+atrophy-determining+gene.">Identification and characterization of a spinal muscular atrophy-determining gene.</a> Cell. 80, 155-165 (1995).</li><li>Schrank, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+survival+motor+neuron+gene,+a+candidate+gene+for+human+spinal+muscular+atrophy,+leads+to+massive+cell+death+in+early+mouse+embryos.">Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 9920-9925 (1997).</li><li>Hsieh-Li, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+spinal+muscular+atrophy.">A mouse model for spinal muscular atrophy.</a> Nat. Genet. 24, 66-70 (2000).</li><li>Monani, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+centromeric+survival+motor+neuron+gene+(SMN2)+rescues+embryonic+lethality+in+Smn(-/-)+mice+and+results+in+a+mouse+with+spinal+muscular+atrophy.">The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy.</a> Hum. Mol. Genet. 9, 333-339 (2000).</li><li>Sleigh, J. N., Gillingwater, T. H., Talbot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+mouse+models+to+understanding+the+pathogenesis+of+spinal+muscular+atrophy.">The contribution of mouse models to understanding the pathogenesis of spinal muscular atrophy.</a> Dis. Model. Mech. 4, 457-467 (2011).</li><li>Durbeej, M., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophies+involving+the+dystrophin-glycoprotein+complex:+an+overview+of+current+mouse+models.">Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models.</a> 12, 349-361 (2002).</li><li>Schnabel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience:+Standard+model.">Neuroscience: Standard model.</a> Nature. 454, 682-685 (2008).</li><li>Benatar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+treatment+trials+in+the+SOD1+mouse+and+in+human+ALS.">Lost in translation: treatment trials in the SOD1 mouse and in human ALS.</a> Neurobiol. Dis. 26, 1-13 (2007).</li><li>Dorchies, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+innervation+and+differentiation+of+X-linked+myotubular+myopathy+muscle+cells+in+a+nerve-muscle+coculture+system.">Normal innervation and differentiation of X-linked myotubular myopathy muscle cells in a nerve-muscle coculture system.</a> Neuromuscul. Disord. 11, 736-746 (2001).</li><li>McFerrin, J., Engel, W. K., Askanas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+innervation+of+cultured+human+muscle+overexpressing+betaAPP+experimentally+and+genetically:+relevance+to+inclusion-body+myopathies.">Impaired innervation of cultured human muscle overexpressing betaAPP experimentally and genetically: relevance to inclusion-body myopathies.</a> Neuroreport. 9, 3201-3205 (1998).</li></ol>

No tenemos nada que revelar.

Una herramienta fisiológica in vitro para estudiar la función de las células musculares en el contexto normal y lo patológico es de mayor interés para los myologists, debido a cultivos de células musculares no suelen recapitular la importancia de las células y las conexiones múltiples de tipo celular. La adición de las neuronas motoras purificadas a las células musculares no es suficiente para lograr una unidad de motor funcional ya que la presencia de células de Schwann se requiere para la inervaci?...

<table border="1"><tbody><tr><td> Nombre de reactivo </td><td> Empresa </td><td> Número de catálogo </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> Medio 199 </td><td> Gibco / Invitrogen </td><td> 31153 </td></tr><tr><td> Suero Bovino Fetal </td><td> Fetal Clone Perbio </td><td> SH30066.03 </td></tr><tr><td> La insulina </td><td> Sigma </td><td> I9278 </td></tr><tr><td> EGF humano </td><td> Sigma </td><td> E9644 </td></tr><tr><td> Humanos FGF </td><td> SiGMA </td><td> F0291 </td></tr><tr><td> La penicilina / estreptomicina solución </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

Las células musculares primarias cultivaron en monocapa aneurally raramente contraen espontáneamente porque, en ausencia de un componente de nervio, la diferenciación celular y el motor se limita la estimulación de neuronas que falta 1. Estas limitaciones dificultan el estudio in vitro en el de muchas enfermedades neuromusculares en las células musculares cultivadas. Es importante destacar que las limitaciones experimentales de células monocapa, músculo cultivadas pueden ser superados por la inervación funcional de miofibras con explantes de médula espinal en co-cultivos. Aquí, se muestran los diferentes pasos necesarios para lograr una inervación eficiente, adecuada de las células musculares primarios, que conduce a completar la diferenciación y la contracción de fibra de acuerdo con el método desarrollado por Askanas 2. Para ello, las células musculares son co-cultivadas con explantes de la médula espinal de embriones de rata en el ED 13,5, con los ganglios de la raíz dorsal todavía unido a las rebanadas de la médula espinal. Después de unos días, el músculo fibros comenzará a contraerse y, eventualmente, convertirse en la cruz-estriado a través de inervación por neuritas funcionales que se proyectan desde los explantes de la médula espinal que se conectan a las células musculares. Esta estructura puede mantenerse durante muchos meses, simplemente por intercambio regular del medio de cultivo. Las aplicaciones de esta herramienta de valor incalculable son numerosas, ya que representa un modelo funcional para realizar análisis multidisciplinarios de desarrollo de los músculos humanos y la inervación. De hecho, una completa instalación de novo unión neuromuscular se produce en un plato de cultivo, permitiendo una fácil medición de muchos parámetros en cada paso, en un contexto fundamental y fisiológicas. Sólo para citar algunos ejemplos, genómica y / o estudios de proteómica se puede realizar directamente en la co-culturas. Además, los efectos de pre y post-sináptica puede ser específica y se evaluó por separado en la unión neuromuscular, debido a que ambos componentes provienen de diferentes especies,rata y humano, respectivamente. El nervio-músculo co-cultivo también puede realizarse con células musculares humanas aisladas de pacientes que sufren de enfermedades neuromusculares músculo o 3, y por lo tanto puede ser utilizado como una herramienta de detección para los fármacos candidatos. Finalmente, ningún equipo especial, pero una instalación normal BSL2 se necesita para reproducir una unidad de motor funcional en una placa de cultivo. Este método, por lo que es valioso tanto para el músculo, así como las comunidades de investigación neuromusculares para estudios fisiológicos y mecanicista de la función neuromuscular, en un contexto normal y la enfermedad.

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

Las células cultivadas del músculo son un modelo inadecuado para recapitular músculo inervado<em&gt; En vivo</em&gt;. Una unidad motora funcional puede ser reproducida<em&gt; In vitro</em&gt; Por la inervación de las células humanas diferenciadas musculares primarias, utilizando explantes de embriones de rata de la médula espinal. En este artículo se describe cómo co-cultivos de explantes de la médula espinal y las células musculares se han establecido.

Una unidad motora funcional en la placa de cultivo: Co-cultivo de explantes de la médula espinal y las células musculares

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

a-functional-motor-unit-culture-dish-co-culture-spinal-cord-explants

Research

JoVE Journal

Neuroscience

15.2K vistas.  University of Basel. Las células cultivadas del músculo son un modelo inadecuado para recapitular músculo inervado En vivo. Una unidad motora funcional puede ser reproducida In vitro Por la inervación de las células humanas diferenciadas musculares primarias, utilizando explantes de embriones de rata de la médula espinal. En este artículo se describe cómo co-cultivos de explantes de la médula espinal y las células musculares se han establecido.

Video: 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.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

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

Investigación

Neurociencias

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.

La disección del ganglio y Cultura polluelo Statoacoustic y explantes médula espinal en geles de colágeno para ensayos neurite

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.

Embriones de pollo de la Médula Espinal Slice Cultura Protocolo

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.

Análisis de Desarrollo del diente Germ Inervación Uso de dispositivos de microfluidos Co-cultura

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.

El pez cebra<em&gt; In Situ</em&gt; Médula Espinal Preparación para Registros electrofisiológicos de Spinal Sensory y neuronas motoras

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.

Laminectomía e implantación de la ventana de la médula espinal en el ratón

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.

Identificación de marcadores de superficie celular de células madre neuronales primarias y células progenitoras por etiquetado metabólico de sialoglicano

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.

Inyección directa de un Vector lentivirales destaca múltiples caminos del Motor en la médula espinal de rata

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.

Conversión de células madre pluripotentes inducidas por humanos (IPSC) en neuronas motoras craneales y espinales funcionales usando vectores 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.

Entrega intra-arterial de células madre neuronales a la rata y el cerebro del ratón: aplicación a la isquemia cerebral

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