Este trabajo fue apoyado en parte por los Institutos Nacionales de Salud: HD054347 y NS063997-01 de VLS. Este trabajo también fue apoyado en parte por el Fondo Empire State de células madre a través del Estado de Nueva York Departamento de Salud Contrato # C024324 de VLS. Las opiniones expresadas aquí son de exclusiva responsabilidad del autor y no reflejan necesariamente el parecer de la Junta del Empire State de Células Madre, el Estado de Nueva York Departamento de Salud, o el Estado de Nueva York. VLS es un Doris Duke destinatario científico clínico Premio del Desarrollo. También agradecemos el profesor Timothy Vartanian por su don de Anti-O1, O4 Anti-anticuerpos.

1. Preparación de soluciones y materiales para la disección y el mantenimiento de la cultura de células madre neurales <ol><li> Prepare 100 ml medio de disección (KNOCKOUT DMEM/F12, Invitogen) antes de tiempo y refrigere. </li><li> Prepare100 ml medio de cultivo (Stem Pro NSC SFM, Invitrogen) y mantener a 37 ° C en un baño de agua. </li><li> Prepare la celda del punto de congelación medio (KNOCKOUT DMEM/F12 10% de SFB + 5% de DMSO) para la crioconservación a largo plazo de las células. </li><li> Si lo desea, prepare un 4% de paraformaldehído (PFA) para la fijación del tejido. </li><li> Pinzas esterilizadas, autoclave y hojas de bisturí con mango se utiliza para la disección. </li><li> Dejar de lado una pistola pipeta, 10 ml pipetas de transferencia, y 40 M de células colador (BD Falcon 352 340) para la disociación. </li><li> Ponga a un lado varios platos de 10 cm de la cultura (BD) para la disección, 50 tubos de centrifugación ml (BD) para la disociación de 1,5 ml tubos de centrifugación para el almacenamiento de tejidos y el congelamiento (BD) para congelar células. </li></ol> 2. Aislar las células madre neurales del cerebro fetal humano <ol><li> La recolección de tejido viable inmediatamente después de la terminación del feto es vital para el éxito del procedimiento. Para los procedimientos electivos, el tiempo para obtener las muestras se pueden arreglar de antemano con el fin de minimizar el tiempo después de la muerte fetal. Los productos de la concepción se recogen dentro de las 2 horas después del procedimiento, pero lo ideal se puede lograr en los procedimientos electivos en cuestión de minutos. Los tejidos del feto con frecuencia se fragmentan. Sin embargo, en general, una parte importante del cerebro permanece intacto para la identificación visual. Limitaciones de la edad gestacional (EG) son determinados por la ley estatutaria, pero se han realizado con este protocolo entre 18-22 semanas de gestación. </li><li> Cerebro del feto se coloca en un plato de Petri que contiene 10 ml de hielo frío KNOCKOUT DMEM/F12 solución. Identificar las diferentes partes de la corteza de puntos de referencia anatómicos. Los límites de la corteza frontal y parieto-occipital están orientadas por la intersección de la extrapolación surco central y la cisura de Silvio. Disecar el tejido de la corteza frontal anterior al surco central ya lo largo de la frontera de la cisura de Silvio con cuchillas quirúrgicas, asegurándose de mantener el ventrículo intacto y sin daños. </li><li> Eliminar cualquier residuo de sangre y las meninges del bloque separado de la corteza frontal. Si la muestra es de suficiente calidad, es ideal para diseccionar el bloque en varias muestras más pequeñas para diversos fines: secciones (fija en el 4% de paraformaldehído, PFA) y la proteína / ARNm ensayos (rápido congelado en -80 ° C). </li><li> Transferencia del bloque cerebro selecciona a un tubo de centrífuga de 50 ml, y añadir helada KNOCKOUT DMEM/F12 solución en aproximadamente 3 veces el volumen del tejido. Suavemente disociar el tejido con la pipeta mecánica con una pipeta de transferencia de 10 ml hasta que todos los tejidos se fragmenta (por lo general 20-30 horas), y luego filtrar las células a través de un filtro de 40 micras de células (BD Falcon 352 340) para obtener una sola célula o cerca de suspensión. </li><li> Se centrifuga la suspensión celular a 2000 rpm y la temperatura ambiente durante 5 minutos, resuspender el botón celular en 10 ml de medio de cultivo fresco caliente (Stem Pro NSC SFM), y contar el número de células con un hemocitómetro. </li><li> Añadir 5 ml de medio de cultivo caliente en frascos cada 25 cm 2 cultura, y la transferencia de 2X10 6 celdas a cada frasco. Cultivos se mantienen en un 37 ° C / 5% CO 2 incubadora durante 1 semana antes del análisis. Cambiar la mitad de la media una vez por semana para que las culturas más o experimentos. </li></ol> 3. Manipulaton de las células madre neuronales para la caracterización adicional o experimentaciones <ol><li> Neuroesferas generalmente se forman en 1 a 2 semanas bajo las condiciones de cultivo se recomienda con un diámetro de NSC oscilan entre los 200 y 400 micras. Neuroesferas en esta etapa se puede disociar con 0,2 g / L de EDTA libre de calcio y magnesio medio Hanks (Hanks) a 37 ° C durante 15 minutos para obtener células individuales. Suspensión de células se centrifugan a 2000 rpm, lavadas en agua dulce Hanks, y replated en medio de cultivo de abrigo para la subcultura. </li><li> Para iniciar la diferenciación, las células disociadas se colocan en poly-D-lysine/laminin un cubreobjetos recubiertos con una densidad de 1x10 5 células por cubreobjetos (24mmX24mm). La diferenciación de oligodendrocitos se logra mediante el mantenimiento de las células en KNOCKOUT DMEM/F12 (Invitrogen, principal, MD) 2% B27 (50X, Invitrogen, principal, MD) 10 ng / ml bFGF 100 ng / ml + SHH 10ng/ml PDGF-AA dos días, luego de cambiar al mismo medio sin los factores de crecimiento por otros 5 días. Diferenciación neuronal se logra mediante el mantenimiento de las células en KNOCKOUT DMEM/F12 2% B27 (50X) durante 7 días. La diferenciación de astrocitos se realiza el cultivo de células en un KNOCKOUT DMEM/F12% de SFB durante una semana. </li><li> La transfección de células madre neurales con los genes se puede hacer con células disociadas de las neuroesferas preformada. En este sentido, mostraron que las células madre neurales transfectadas con EGFP-C1 de la electroporación. La electroporación de EGFP-C1 construcción se realizó mediante kits Amaxa Nucleofector de células madre neurales (VPG-1004) con Amaxa dispositivo Nucleofector (Lonza AAD-1001), siguiendo las instrucciones de la empresa. En resumen, 5 mg de ADN con 1 x 10 6 células se mezclan con 100 l medio de transfección, y después de la electroporación de pulso, se resuspendieron las células en las células madre neurales mantener medio de cultivo adicional. La diferenciación de las células transfectadas fueron procesados ​​con la disociación de los días neuroesferas 3-4 después de la electroporación, siguiendo los mismos procedimientos descritos en el paso 3.2. </li></ol> 4. Congelar las células madre neurales y las subculturas <ol><li> Suspensiones de células disociadas se centrifugan y se resuspenden en medio de congelación con una concentración de 1x10 7 células / frasco / ml. Poco a poco congelar las células a -20 ° C, -80 ° C, entonces la transferencia de nitrógeno líquido para su almacenamiento a largo plazo. </li><li> Las células se descongelan rápido con 37 ° C baño de agua caliente y resuspendido en DMEM/F12 10% de suero de un lavado, se centrifuga para eliminar el medio de congelación, y resuspendido en el medio de cultivo caliente. </li></ol> 5. Los resultados representativos: Las células madre neurales de un feto normal de 18 semanas de edad gestacional se cultivaron siguiendo los métodos descritos y neuroesferas se puede ver después de una semana con todo, bordes suaves y de tamaño bastante homogénea (Fig.2a). Estos neuroesferas pueden ser transfectadas con EGFP-C1 o de otras construcciones y seguir bajo el microscopio de fluorescencia (Fig.2b,). Neuroesferas se establecieron entonces disocian con EDTA y se sembraron las células dispersas en cubreobjetos recubiertos. Células diferenciadas en los respectivos protocolos fueron fijadas con 4% parafamaldehyde, y teñidas con marcadores de diferentes tipo de células específicas. Multipotencialidad se observa con expresión de marcadores indicativos de las neuronas (Fig2C, D, rodamina) astrocitos (Fig2E, F, rodamina) y oligodendrocitos (Fig2G, H, rodamina). Las células no sometido a la electroporación de EGFP se diferencian también en diferentes tipos de células y se tiñen con marcadores de células específicas diferentes. Multipotencialidad se observa con expresión de marcadores indicativos de las neuronas (Fig3A, B, fluoresceína) astrocitos (Fig3C, rodamina y Fig3D, fluoresceína) y oligodendrocitos (Fig3E, F, rodamina). <img src="/files/ftp_upload/2681/2681fig1.jpg" alt="Figura 1"/> Figura 1. Esquema del procedimiento experimental para aislar las células madre neurales del cerebro fetal humano descartados <img src="/files/ftp_upload/2681/2681fig2.jpg" alt="Figura 2"/> Figura 2. Indiferenciadas y diferenciadas las células neuronales humanas propagado in vitro. (A) neuroesferas se muestran bajo el microscopio de contraste de fase muestran bordes suaves, redondos y un rápido crecimiento después de la cultura durante más de una semana. (B) introducción de diversos plásmidos y las construcciones se puede lograr a través de la transfección. Tres días después de EGFP-C1 transfección, las células múltiples muestran una expresión de la proteína fluorescente verde como se ve en la inmunotinción fluoresceína y microscopía de fluorescencia. EGFP-C1 neuroesferas transfectadas no están asociadas y diferenciadas en condiciones diferentes a las neuronas (C, D), los astrocitos (E, F) y oligodendrocitos (G, H) y visto en la fluorescencia de rodamina. Al mismo tiempo, las células transfectadas EGFP positivos se presentan en fluoresceína fluorescencia. Las células transfectadas (cabezas flecha blanca) son indistinguibles de las células no transfectadas (flechas blancas). Los núcleos celulares se tiñen con Hoechst33342. Las barras de escala de 200 m para A, 100 micras para B y 25 micras de CH. <img src="/files/ftp_upload/2681/2681fig3.jpg" alt="Figura 3"/> Figura 3. Neuroesferas sin transfección se disocian y diferenciada en diferentes condiciones en las neuronas (A, B, fluoresceína), los astrocitos (C, rodamina, D, fluoresceína) y oligodendrocitos (E, F, rodamina). Los núcleos celulares se tiñen con Hoechst33342. Las barras de escala son de 25 micras para la AF.

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Existen varios enfoques hacia el cultivo de tejidos frescos y la producción de líneas celulares humanas. Históricamente, el tejido fresco ha sido cosechados y cultivados de inmediato para generar diversos tipos de células en el sistema nervioso central. Esta estrategia, sin embargo está claramente limitado por el número de muestras que se pueden obtener, que en el caso de muestras humanas, suele ser bastante pequeña. Teniendo en cuenta el grado mínimo de la manipulación, las células neuronales cultivadas reci?...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> KNOCKOUT DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td> Disociación medio </td></tr><tr><td> Madre Pro NSC SFM </td><td> Invitrogen </td><td> A10509-01 </td><td> Medio de cultivo </td></tr><tr><td> De suero fetal bovino </td><td> Invitrogen </td><td> 10091-148 </td><td> Medio congelados </td></tr><tr><td> Hanks solución (-Ca 2 +, Mg 2 +) </td><td> Invitrogen </td><td> 14175-095 </td><td> Disociación medio </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D2650 </td><td> Medio congelados </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> 431788 </td><td> Disociación medio </td></tr><tr><td> Paraformaldehído </td><td> Sigma-Aldrich </td><td> 158127 </td><td> La fijación de una solución </td></tr><tr><td> bFGF </td><td> I + D </td><td> 234-FSE </td><td> Diferenciación medio </td></tr><tr><td> SHH </td><td> I + D </td><td> 1845-SH </td><td> Diferenciación medio </td></tr><tr><td> PDGF-AA </td><td> I + D </td><td> 221-AA </td><td> Diferenciación medio </td></tr><tr><td> B27 </td><td> Invitrogen </td><td> 17504-044 </td><td> Diferenciación medio </td></tr><tr><td> Ratón anti-MAP2 </td><td> Sigma-Aldrich </td><td> M2320 </td><td> 1:200 </td></tr><tr><td> De conejo anti-DCX </td><td> Señalización celular </td><td> 4604s </td><td> 1:200 </td></tr><tr><td> De conejo anti-GFAP </td><td> Dako </td><td> Z0334 </td><td> 1:200 </td></tr><tr><td> De conejo anti-S100B </td><td> Dako </td><td> Z0311 </td><td> 1:200 </td></tr><tr><td> De conejo anti-O1 </td><td> dones del profesor Timothy Vartanian * </td><td></td><td> 01:50 </td></tr><tr><td> De conejo anti-O4 </td><td> Regalos del profesor Timothy Vartanian * </td><td></td><td> 01:50 </td></tr><tr><td> 40μm de células colador </td><td> BD Falcon </td><td> 352340 </td><td></td></tr></tbody></table> * Timothy Vartanian, MD, PhD, Departamento de Neurología y Neurociencia de la Weill Cornell Medical College, Nueva York, EE.UU.

generation of neural stem cells from discarded human fetal cortical tissue

Las células madre neurales (NSC) residen en la zona ventricular neuroepitelio durante el desarrollo de la placa cortical. Estos primeros progenitores en última instancia, dan lugar a los progenitores intermedios y más tarde, los diferentes subtipos de células neuronales y gliales que forman la corteza cerebral. La capacidad de generar y ampliar NSCs humanos (los llamados neuroesferas) a partir de tejido fetal normal descarta proporciona un medio con el cual estudiar directamente los aspectos funcionales de la normalidad del desarrollo humano NSC 1.5. Este enfoque también puede ser dirigido hacia la generación de NSCs de trastornos neurológicos conocidos, ello se traduzca en la oportunidad de identificar los procesos de enfermedad que altera la proliferación de progenitores, la migración y la diferenciación de 6-9. Nos hemos centrado en la identificación de los mecanismos patológicos en humanos NSCs síndrome de Down que pueden contribuir a acelerar el fenotipo de la enfermedad de Alzheimer 10,11. Ni en vivo ni in vitro modelos de ratón se puede replicar el repertorio idéntico de genes localizados en el cromosoma humano 21. Aquí se utiliza un método sencillo y fiable para aislar el síndrome de Down NSCs de corteza fetal humano abortado y crecer en cultivo. La metodología ofrece aspectos específicos de la recolección de los tejidos, la disección con puntos de referencia anatómicos limitado, separación celular, placas y pases de NSCs humanos. También proporcionamos algunos protocolos básicos para inducir la diferenciación de las NSCs humanos en subtipos de células más selectivo.

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Un método sencillo y fiable en el aislamiento y cultivo de células madre neurales en humanos descartados tejido cortical fetal se describe. Cultivos derivados de enfermedades neurológicas conocidas humanos pueden ser utilizados para la caracterización de los patológicos procesos celulares y moleculares, así como ofrecer una plataforma para evaluar la eficacia farmacológica.

Generación de las células troncales nerviosas de desechados Humanos tejido cortical fetal

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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Neuroscience

15.4K vistas.  Beth Israel Deaconess Medical Center. Un método sencillo y fiable en el aislamiento y cultivo de células madre neurales en humanos descartados tejido cortical fetal se describe. Cultivos derivados de enfermedades neurológicas conocidas humanos pueden ser utilizados para la caracterización de los patológicos procesos celulares y moleculares, así como ofrecer una plataforma para evaluar la eficacia farmacológica.

Video: Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural formadoras de colonias ensayo de células: un ensayo para discriminar Bona Fide células troncales nerviosas de células progenitoras neurales

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Investigación

Neurociencias

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Aislamiento y cultivo de células troncales de la cresta neural de los folículos del cabello humano

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

El ensayo neuroblasto: un ensayo para la generación y enriquecimiento de las células progenitoras neuronales de la diferenciación de la progenie de células madre neurales Usando citometría de flujo

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

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 ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Desarrollo de un protocolo de refinado para Trans-escleral transplante Subretinal de células epiteliales del pigmento retiniano humano a ojos de rata

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Sencilla generación de una cultura de alto rendimiento de las neuronas inducidas de fibroblastos de piel humana adulta

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Grabaciones simultáneas de potenciales de campo Local Cortical, Electrocardiograma, electromiograma y respirar a ritmo de una rata libremente móvil

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...