Este trabajo fue financiado por la ANR (Agence Nationale de la Recherche), AFM (Asociación Francesa contra las Miopatías), FEDER en PACA y Irme (Institut de Recherche sur la Moelle épinière et l&#39;Encéphale). Nosotros agradecemos a Marie Pierre Blanchard (Jean Roche Institute) por su ayuda eficiente durante la grabación de lapso de tiempo.

1. Colección de mucosa olfatoria en ratas <ol><li> Empezar por la elaboración de tres placas de Petri de 35 mm llena de DMEM / HAM F12 medio de cultivo en una campana de cultivo limpio. </li><li> Cualquier método de eutanasia debe ser aprobado previamente por el cuidado de animales de la institución y el uso comisión y llevado a cabo por personal cualificado. Después de la rata ha entrado en una anestesia profunda con el sodio pentobarbital formas inyectables o de otro tipo de anestesia, como la ketamina / xilazina, decapitar y quitar la piel. Anestésicos inhalados deben ser evitados. Adecuación de la anestesia será evaluada por una pizca dedo antes de la decapitación. Retire la mandíbula inferior con unas tijeras y con la ayuda de una gubia, eliminar los músculos faciales en ambos lados. </li><li> A partir de la parte posterior de los incisivos, retire con una gubia del hueso que cubre la cavidad nasal, de un lado a la vez. Los cornetes olfativa entrar en la vista como el naranja / marrón órganos situados en la parte posterior de la nariz. </li><li> Delicadamente descartar los cornetes con una pinza. Usando una aguja de calibre 26, aislar la mucosa olfatoria tendido en el tabique al cortar el tejido a lo largo de tres líneas: el arco de la placa perpendicular, la lámina cribiforme y el techo de la cavidad nasal. </li><li> Recoger las biopsias de ambos lados y la transferencia en un DMEM / HAM F12 lleno de placa de Petri. Este procedimiento no debería tardar más de 10 minutos de la eutanasia inicio. </li><li> Ahora, con el fin de eliminar la mucosidad, la transferencia de las biopsias de dos veces en medio lleno de placas de Petri. </li></ol> 2. Colección de mucosa olfatoria en los seres humanos <ol><li> Este procedimiento debe ser realizado por un Oído, nariz y garganta (ENT), cirujano, de acuerdo con las disposiciones pertinentes de ética local del comité (s), y todos los pacientes ambulatorios deben firmar un formulario de consentimiento informado. </li><li> El uso de un 0 ° o 30 ° endoscopio rígido (4 mm de diámetro), inspeccione ambas cavidades nasales y evaluar la presencia putativa de pólipos o de cualquier lesión inflamatoria. Elige la mejor cavidad nasal, teniendo en cuenta la desviación del tabique. </li><li> Utilizando un aplicador de algodón, aplicar un anestésico local, como la lidocaína con epinefrina, durante 10 minutos. </li><li> Con unas pinzas etmoidal throughcut, se recoge una biopsia de dos milímetros cuadrados, ya sea en la raíz de la cara medial del cornete medio o en el tabique en la zona dorsomedial. </li><li> El olfato biopsia se transfiere, utilizando una aguja estéril, en un tubo estéril de 2 ml llena de 1 ml de DMEM / HAM F12. Punta del tubo hacia abajo para asegurarse de que la biopsia se encuentra inmerso en el medio de cultivo. </li><li> Inserte el tubo en un contenedor refrigerado y transporte al laboratorio de investigación. En esta etapa, la biopsia se puede utilizar por sí mismo para estudios comparados molecular centrado en enfermedades específicas del cerebro o procesados ​​para la generación de células madre. </li></ol> 3. Aislamiento de células madre olfativa de humano y de rata Mucosa <ol><li> Lave las biopsias en DMEM / HAM F12. Incubar las biopsias en una placa de Petri llena con 1 ml de solución dispasa II (2,4 UI / ml), durante 1 hora a 37 ° C. </li><li> A continuación, en un microscopio de luz invertida con una difracción, el epitelio olfativo se retira de la lámina propia subyacente con una espátula de micro. </li><li> El epitelio olfativo es más delgada y se ve transparente sobre un fondo negro en comparación con la lámina propia, que es a rayas de color naranja / marrón. Sobre un fondo blanco, el epitelio se ve gris y la lámina propia, marrón. </li><li> Una vez purificada, la transferencia de la lámina propia en una placa de Petri llena de DMEM / HAM F12. </li><li> Si el tejido es de un roedor, a continuación, cortar la lámina propia en pequeños trozos con dos agujas de calibre 25. Entonces, la transferencia de las piezas a un tubo de 15 ml llena de 1 ml de colagenasa IA. </li><li> En el tubo, usando una pipeta de plástico estéril, disociar el tejido. A continuación, se incuba el tubo durante 10 minutos a 37 ° C. </li><li> Para terminar la disociación, agite con cuidado el tubo y agregar 9 ml de Ca-Mg libre y PBS libre y centrifugar a 200 g durante 5 minutos. </li><li> Resuspender el botón celular en DMEM / HAM F12 medio de cultivo complementado con suero bovino fetal al 10%, los antibióticos y la placa en placas de cultivo de plástico. </li><li> Ahora bien, si el tejido es humano, entonces parte de la lámina propia en 3 a 4 piezas con un grosor de entre 200 a 500 micras. </li><li> Insertar cada tira en su propio plato de 2 cm de diámetro y la cultura cubrir el tejido con hojas estériles 1,3 cm de diámetro cubierta de vidrio. </li><li> A continuación, añadir 500 l de medio de cultivo (DMEM / HAM F12 suplementado con suero bovino fetal al 10% y antibióticos) para cada placa de cultivo. </li><li> Para cualquier tipo de tejido, renovar el medio de cultivo cada 2 ó 3 días. </li><li> De cinco a siete días después, las células madre comienzan a invadir la placa de cultivo y después de dos semanas deben ser confluentes. Cuando se llega a la confluencia, la aprobación y la transferencia de las células a frascos de cultivo. </li></ol> 4. Ámbito de formación y difere neuronalntiation olfativo de las células madre <ol><li> Para generar ámbitos de células madre, se incuban los frascos durante dos horas a 37 ° C, con poli-L-lisina. (TEXTO: 5 g/cm2). </li><li> Placa de las células a una densidad de 16.000 células por centímetro cuadrado en los frascos tratados. </li><li> Cada dos días, se alimentan de las células con 0,2 ml por centímetro cuadrado de medio suplementado (DMEM / HAM F12 suplementado con insulina, transferrina, el selenio (ITS-X, 1%), EGF (50 ng / ml) y FGF2 (50 ng / ml)). </li><li> De dos a cinco días más tarde, recoger esferas flotantes de células y, o bien la placa de re-o se disocian antes de injerto en modelos animales de la terapia celular. </li><li> Para diferenciar las células madre en neuronas olfativas-como las células, las cultivan durante 21 días en un medio que contiene Neurobasal B-27, la penicilina, la estreptomicina, glutamina y glutamato. </li><li> De hacer cambios en el medio cada 3 días. Neuronas como células deben aparecer después de dos a tres semanas. </li></ol> 5. Los resultados representativos: Nasal humano explante, superando las células madre (Figura 2) se dividen rápidamente y se puede llegar a la confluencia en una o dos semanas. Una característica clave de la troncalidad, nestin expresión, fue evaluado (Figura 2B). Cuando se cultiva en poly-L-lisina con un medio de cultivo libre de suero suplementado con EGF (50 ng / ml) y FGF2 (50 ng / ml), las células olfativas madre dan lugar a las esferas (figura 2C). Cuando se cultiva en medio de cultivo que contiene suero recién esferas plateado dar lugar a células que expresan GFAP (~ 50%), las células que expresan la tubulina (~ 10-15%) y O4, que expresan las células (~ 5.2%) 9 (Figuras 2D-F). Sin embargo, el destino de las células derivadas de esfera puede ser modificada. Por ejemplo, cuando se cultivan en un medio de cultivo Neurobasal complementado con B27 y el glutamato, la mayoría de las células madre en neuronas olfativas nasal-como las células que expresan β-tubulina III (Figura 2 G) y MAP2 (Figura 2H). <img src="/files/ftp_upload/2762/2762fig1.jpg" alt="Figura 1"/> Figura 1. Esquema general del experimento. Biopsias de la mucosa olfativa son extirpados de ratas o de la cavidad nasal humana. Los explantes se puede utilizar por sí mismo para estudios comparativos con el objetivo molecular para identificar los biomarcadores en enfermedades del cerebro. Para aislar las células olfativas del tallo, las interacciones entre la lámina propia y el epitelio neuro-se interrumpió con la enzima dispasa II y, después de 45 minutos, el epitelio se retira con una espátula de micro. Las células madre son roedores olfativo más seleccionados por la disociación de la lámina propia con colagenasa IA. Para los tejidos humanos, pedazos de lámina propia olfatoria son cultivadas en cubreobjetos de vidrio hasta superando las células madre invaden todo bien. Después de la proliferación, utilizando un medio de cultivo adecuado, las células olfativas madre pueden generar esferas o diferenciarse en neuronas como las células. Células olfativas madre se pueden utilizar para: i) las enfermedades de la reparación cerebral o un traumatismo, o ii) la identificación de marcadores moleculares de enfermedades del sistema nervioso. Las ilustraciones están hechas con la ayuda de Servier arte médico. <img src="/files/ftp_upload/2762/2762fig2.jpg" alt="Figura 2"/> Figura 2. Cultura y la diferenciación de células madre humanas nasales olfativas. Las células madre humanas que surgen de la lámina propia explante (A) se dividen rápidamente, cuando se cultivan en un medio que contiene suero. Las células madre expresan la troncalidad marcador nestina (B). Cuando se siembran en poli-L-lisina de plástico y se cultivan en un medio de cultivo libre de suero complementado con EGF y FGF2, las células olfativas madre generar ámbitos (C). Esfera células derivadas, cuando se siembran en el medio de cultivo que contiene suero, dan lugar a células que expresan GFAP (~ 50%), las células que expresan la tubulina (~ 10-15%) y O4, que expresan las células (~ 2.5%) 9 (DF). Cuando se cultiva en un medio de cultivo Neurobasal complementado con B27 y el glutamato, que se diferencian en neuronas como células que expresan β-tubulina III (G) y MAP2 (H).

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No hay conflictos de interés declarado.

Las técnicas presentadas aquí hacen los roedores y la mucosa olfativa humana un modelo útil para la investigación clínica sobre las causas de las enfermedades del neurodesarrollo y neurodegenerativos, así como una herramienta para reparar el cerebro patológico o traumatizados. El protocolo es relativamente sencillo y puede ser fácilmente llevados a cabo por un biólogo celular de la experiencia. La tasa de éxito de las técnicas de biopsia y cultivo es alto. Los pasos críticos <ul><li> Para la recogida de la mucosa olfatoria de roedores, se recomienda no exceder un límite de tiempo de 10 minutos entre la eutanasia y la escisión definitiva del tejido olfativo. </li><li> La disociación de la propia lámina de roedores olfativa se logra generalmente después de 10 min. incubación en la colagenasa IA. Si no es así, se recomienda recoger al día siguiente del sobrenadante en el que los bits no disociado de acciones en circulación de la lámina propia, se centrifuga a 200 g y mecánicamente disociar la lámina flotante con una pipeta Pasteur antes de replating las células de un nuevo pozo. El primer pozo que contiene las células ya instalada está llena de medio de cultivo fresco. </li><li> De la lámina propia humana, que es más compacto que el tejido de los roedores, no recomendamos una disociación enzimática. El tejido se corta y cada explante se inserta entre la parte inferior del plato de plástico y un cubreobjetos. Culturas exitosas incluyen explantes cuyo espesor rango 200 a 500 micras. </li></ul> Posibles modificaciones <ul><li> El protocolo actual puede ser ligeramente modificada con el fin de generar neuronas olfativas in vitro. A tal efecto, la neuro-epitelio no se quita y la mucosa olfatoria todo se corta con un helicóptero McIlwain (200 micras de espesor). Cada explante se platea en un plato, parcialmente seco durante una hora y luego rehidratados con medio de cultivo que contienen FCS. Durante el forro días después de la primera, las células epiteliales y mesenquimales surgen de los explantes. Entonces, los progenitores neuronales migrarán en la parte superior de esta capa de células y se diferencian en neuronas. </li><li> Purificación de las células madre olfativa se puede lograr mediante citometría de flujo. Marcadores específicos de superficie puede ser recuperada de la lista publicada en el documento de caracterización 11. </li><li> Para los experimentos de trasplante, es posible utilizar una cepa de ratones cuyas células olfativas madre son las buenas prácticas agrarias-positivas. Esta cepa de rata (Sprague Dawley, eGFP impulsada por el promotor PGK) está disponible en ITERT (Nantes, Francia). Para insertar el gen de la GFP en las células madre olfativa, se utiliza un método basado en la infección por lentivirus. </li><li> Este documento se centra en las células madre olfativa ecto-mesenquimal. Sin embargo, otro tipo de célula de interés, las células olfativas ensheathing, puede purificar a partir del mismo tejido. Se describe un método para su obtención y purificación de 1,17 y se utilizaron las células nasales ensheathing de una fase I / IIa de ensayos clínicos en pacientes parapléjicos 18. </li><li> Como miembro de la superfamilia de las células madre mesenquimales, OE-MSC son capaces, bajo condiciones de cultivo adecuadas, que se diferencian en adipocitos, osteocitos y los miocitos 11. </li></ul> Las futuras aplicaciones <ul><li> Ya hemos utilizado biopsias humanas olfativas para estudiar las anomalías celulares y moleculares en pacientes con autismo, trastorno bipolar, disautonomía familiar, enfermedad de Parkinson, la enfermedad de Alzheimer y la esquizofrenia 2.7. En teoría, todas las enfermedades del cerebro pueden ser estudiados usando biopsias nasales o células madre periféricas olfativo. </li><li> Roedores y las células madre olfativo nasal ya han sido injertados en modelos animales de la amnesia, la enfermedad de Parkinson, daño coclear y traumatismo de la médula espinal 12-16. Cabe la posibilidad de trasplantar estas células en modelos animales de enfermedad de Alzheimer, isquemia cerebral, la esclerosis múltiple. </li></ul>

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td colspan="3"> Colección de la mucosa olfatoria en ratas </td></tr><tr><td> DMEM / HAM F12 </td><td> Invitrogen </td><td> 31331-028 </td></tr><tr><td> Pentobarbital sódico </td><td></td><td></td></tr><tr><td> Gubia </td><td> FST </td><td></td></tr><tr><td> 26 calibre de la aguja </td><td> Terumo </td><td> NN-2613R </td></tr><tr><td> Fórceps </td><td></td><td></td></tr><tr><td colspan="3"> Colección de la mucosa olfatoria en los seres humanos </td></tr><tr><td> Endoscopio rígido </td><td> Karl Storz o Richard Wolf Médico </td><td></td></tr><tr><td> Lidocaína </td><td></td><td></td></tr><tr><td> Epinefrina </td><td></td><td></td></tr><tr><td> Throughcut etmoidal fórceps </td><td> Karl Storz o Richard Wolf Médico </td><td></td></tr><tr><td colspan="3"> Aislamiento de células madre olfativo </td></tr><tr><td> Dispasa II </td><td> Roche </td><td> 10 295 825 001 </td></tr><tr><td> Microscopio de disección </td><td></td><td></td></tr><tr><td> Espátula de micro </td><td> FST </td><td></td></tr><tr><td> Colagenasa IA </td><td> Sigma-Aldrich </td><td> C9891 </td></tr><tr><td> Ca-free/Mg-free PBS </td><td> Invitrogen </td><td> 14190-250 </td></tr><tr><td> Suero fetal bovino </td><td> Invitrogen </td><td> 10270098 </td></tr><tr><td> Cubreobjetos de vidrio </td><td> Knittel Glaser </td><td> 001/35 </td></tr><tr><td colspan="3"> Ámbito de formación y diferenciación neuronal </td></tr><tr><td> Poli-L-lisina </td><td> Sigma-Aldrich </td><td> P-1274 </td></tr><tr><td> La insulina de transferrina selenio (ITS) </td><td> Invitrogen </td><td> 51500056 </td></tr><tr><td> FEAG </td><td> R &amp; D Systems </td><td> 236-EG </td></tr><tr><td> FGF2 </td><td> R &amp; D Systems </td><td> 233-FB </td></tr><tr><td> Medio Neurobasal </td><td> Invitrogen </td><td> 21103-049 </td></tr><tr><td> B-27 libre de suero Suplemento </td><td> Invitrogen </td><td> 17504-044 </td></tr><tr><td> Penicilina / estreptomicina </td><td> Invitrogen </td><td> 15140122 </td></tr><tr><td> Glutamina </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Glutamato </td><td> Sigma-Aldrich </td><td></td></tr></tbody></table>

isolating nasal olfactory stem cells from rodents or humans

La mucosa olfatoria, que se encuentra en la cavidad nasal, se encarga de detectar olores. También es el único tejido nervioso que se encuentra expuesta al ambiente externo y de fácil acceso en todos los individuos que viven. Como resultado, este tejido es único para cualquier persona con el objetivo de identificar las anomalías moleculares en el cerebro patológico o aislar las células madre adultas para la terapia celular. Alteraciones moleculares en las enfermedades cerebrales son estudiados a menudo usando muestras de tejido nervioso recogido post-mortem. Sin embargo, este material tiene numerosas limitaciones. Por el contrario, la mucosa olfativa es de fácil acceso y puede hacer una biopsia de forma segura sin ningún tipo de pérdida del sentido del olfato 1. En consecuencia, la mucosa olfativa ofrece una &quot;ventana abierta&quot; en el adulto humano a través del cual se puede estudiar el desarrollo (autismo, la esquizofrenia, por ejemplo,) 2-4 o neurodegenerativas (como Parkinson, Alzheimer) enfermedades 4,5. Mucosa olfatoria se puede utilizar ya sea para estudios comparativos molecular 4,6 o en experimentos in vitro sobre la neurogénesis 3,7. El epitelio olfativo es también un tejido nervioso que produce nuevas neuronas cada día para reemplazar a aquellos que están dañados por la contaminación, bacterias de infecciones virales. Esta neurogénesis permanente se sustenta por los progenitores, sino también las células madre que residen dentro de los dos compartimientos de la mucosa, es decir, la neuroepithelium y el 10.8 lámina propia subyacente. Recientemente hemos desarrollado un método para purificar las células madre adultas ubicados en la lámina propia y, después de haber demostrado que están estrechamente relacionadas con las células mesenquimales de la médula ósea (BM-MSC), que nombró olfativa ecto-mesenquimal de células madre (OE- MSC) 11. Curiosamente, en comparación con el BM-MSC, OE-MSC mostrar una tasa de proliferación de alto, un clonogenicidad elevado y una inclinación de diferenciarse en células neuronales. Nos aprovechamos de estas características para llevar a cabo estudios específicos para dar a conocer nuevos genes candidatos en la esquizofrenia y la enfermedad de Parkinson 4. Nosotros y otros han demostrado también que la OE-MSC son candidatos prometedores para la terapia celular, después de un traumatismo de la médula espinal 12,13, un daño coclear en un 14 o modelos animales de enfermedad de Parkinson 15 o amnesia 16. En este estudio, se presentan los métodos de biopsia de mucosa olfatoria en ratas y humanos. Después de la recolección, la lámina propia es enzimáticamente separada del epitelio y las células madre se purifican mediante un enzimáticos o un método no enzimático. Purificada de células olfativas madre pueden ser cultivadas en grandes cantidades y en bancos en nitrógeno líquido o inducidas a formar esferas o diferenciarse en células neuronales. Estas células madre también se pueden utilizar para ómicas comparativa (genómica, transcriptómica, epigenómico, proteómica) los estudios.

Watch this Scientific Journal Video about Isolating Nasal Olfactory Stem Cells from Rodents or Humans at JoVE.com

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Se describe aquí un método para la biopsia de mucosa olfatoria de ratas y humanos cavidades nasales. Estas biopsias se pueden utilizar tanto para la identificación de anomalías moleculares en las enfermedades cerebrales o el aislamiento de células madre adultas multipotentes que pueden ser utilizados para el trasplante de células en modelos animales de trauma cerebral / enfermedad

Aislar las células madre olfativo nasal de los roedores o los seres humanos

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

isolating-nasal-olfactory-stem-cells-from-rodents-or-humans

Research

JoVE Journal

Neuroscience

30.5K vistas.  Aix Marseille University. Se describe aquí un método para la biopsia de mucosa olfatoria de ratas y humanos cavidades nasales. Estas biopsias se pueden utilizar tanto para la identificación de anomalías moleculares en las enfermedades cerebrales o el aislamiento de células madre adultas multipotentes que pueden ser utilizados para el trasplante de células en modelos animales de trauma cerebral / enfermedad

Video: Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Las mediciones electrofisiológicas de un sistema olfativo Polilla

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Investigación

Neurociencias

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Aislar las células que expresan LacZ de tejidos de ratón del oído interno mediante citometría de flujo

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

La expansión de las células madre embrionarias y adultas neurales por<em&gt; In Utero</em&gt; La electroporación o inyección estereotáxica Viral

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Aislamiento y cultivo de células de la cresta neural de embriones murinos del Tubo Neural

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

Un método de expansión de GMPc aplicable para agregados de Neural humano madre y células progenitoras derivadas de células madre pluripotentes o tejido cerebral fetal

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivación-Feeder libres de Células Progenitoras de la cresta neural Pluripotentes Células Madre

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dirigida dopaminérgica Neurona La diferenciación de las células madre pluripotentes humanas

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelado astrocitoma Patogénesis<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Uso cortical astrocitos o células troncales nerviosas de ratones condicionales, Genéticamente Manipulados

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Las neuronas olfativas obtenidos por medio de biopsia nasal combinado con microdisección por láser captura: un enfoque potencial para el Estudio de la respuesta al tratamiento en los trastornos mentales

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Diferenciación neuronal de las células madre embrionarias del ratón in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...