Este trabajo fue apoyado por becas del Centro de Florida para la Investigación de Tumores Cerebrales, Preston A. Wells Jr. Centro de Terapia tumor cerebral.

1. La acumulación de tejido GBM primaria <ol><li> Un glioblastoma mutliforme (GBM) del tumor se obtiene de un paciente diagnosticado con el cáncer y someterse a una cirugía. </li><li> Se deben hacer arreglos con el equipo de neurocirugía. El neurocirujano lugar del tumor resecado GBM en un tubo de tamaño adecuado que contiene células madre neurales (NSC) medio basal suplementado con antibióticos 10-15% (Peniciline / estreptomicina). Medio frío se debe proporcionar a la sala de operaciones de laboratorio. Por otra parte, el frío HEPES buffer medio esencial mínimo (HEM) o tampón fosfato salino (PBS) con alta concentración de antibióticos también se pueden utilizar para este propósito. </li><li> El tejido del tumor resecado se entrega al laboratorio en hielo y se coloca bajo el capó. </li></ol> 2. La disociación de un tumor primario en una sola suspensión celular <ol><li> El exceso del medio original / PBS se retira el tubo de halcón y la muestra se lava 2-3 veces con 5-10ml de PBS / NSC medio de base para extraer la sangre y los escombros. El PBS / medio se elimina y el tejido tumoral GBM se coloca en una placa de Petri. </li><li> El tejido se corta en trozos pequeños y picados con una hoja de bisturí N º 10 en pequeños pedazos para aumentar la superficie para el proceso de tripsina. Picado puede tomar de 1-3 minutos, dependiendo del tamaño del tumor. </li><li> El tejido picado se trypsinized en 3-5ml de pre-calentado 0,05% de tripsina-EDTA durante 10-15 minutos en un baño de agua a 37 ° C. Una pipeta electrónica se utiliza para transferir las piezas tumor pequeño y tripsina en un tubo de 15 ml Falcon. </li><li> Un volumen igual de inhibidor de tripsina de soja se agrega para detener la reacción enzimática con tripsina después del período de incubación. </li><li> Inactivación de la tripsina se garantiza con la pipeta de la suspensión hacia arriba y abajo varias veces. Luego, la suspensión se sedimentan por centrifugación a 800rpm (110 g) durante 5 minutos. </li><li> El sobrenadante se descarta y las piezas de tejido se resuspendió en 1 ml de agua estéril NSC basal media. Los grupos se disocian pipeteando suavemente arriba y abajo (3-7 veces) hasta que una suave suspensión lechosa sola célula se logra. El número de pasos de pipeteo depende directamente del tamaño de las partículas en el tejido picado. Disociación mecánica prolongada y vigorosa se debe evitar, ya que podría resultar en la muerte celular y una reducción en la formación de la esfera. </li><li> Para eliminar sin disociar las piezas y los escombros, 10-15 ml de medio basal se añade al tubo y la suspensión celular se filtra a través de un colador 40 micras de células en un tubo de 50 ml. </li><li> Se filtró la suspensión se centrifuga a 800rpm (110 g) durante 5 minutos. El sobrenadante se descarta después. </li><li> Pastillas de células son luego resuspendido en 1-2 ml de medio completo NSC para el recuento de células. </li></ol> 3. Recuento de células y Revestimiento <ol><li> 10 l de la suspensión celular se añade a 90 L de Trypan 0,04% azul en un tubo eppendorf 1 ml. Nota: otras células apropiadas diluciónciones también se puede utilizar. </li><li> Pipeta de arriba y abajo para mezclar la suspensión. 10 l de las células / mezcla de azul tripán se transfiere a hemocitómetro con el fin de contar con la densidad celular. </li><li> Las células se sembraron en medio completo NSC (una mezcla de medio basal NSC NSC y complementar la proliferación en una proporción de 9:01), complementado con 20ng/ml EGF, bFGF 10ng/ml y 1μl/ml del 0,2% con heparina (2μg/ml) en apropiado vasos de cultivo de tejidos. 5 ml, 20 y 40 de medio se utiliza para frascos T25, T80 y T175, respectivamente. Los antibióticos pueden ser añadidos al medio a una concentración de 1:100 para disminuir la posibilidad de contaminación. </li><li> El matraz se coloca en una incubadora a 37 ° C y 5% CO 2. </li></ol> 4. Pases y ampliación de las esferas GBM derivados: <ol><li> Cuando las neuroesferas alcanzado un tamaño promedio de 150 a 200 micras de diámetro, la cultura está listo para la subcultura. El contenido de cada matraz se retira y se coloca en un TISS tamaño estéril apropiadatubo ue la cultura, y se centrifuga a 800 rpm (110 g) durante 5 min a temperatura ambiente. </li><li> Se elimina el sobrenadante y el sedimento se resuspende en 1 ml de 0,05% de tripsina-EDTA. </li><li> Para lograr un óptimo tripsinización, la suspensión de células se incubaron a 37 º C en un baño de agua durante 2-3 min. Para detener la actividad de la tripsina un volumen igual de inhibidor de tripsina de soja se añade a la suspensión celular y la suspensión celular es ligeramente pipeta hacia arriba y abajo. </li><li> La suspensión celular se centrifuga a 800 rpm (110 g) durante 5 min. Luego, se retira el sobrenadante y las células se resuspenden en 1 ml de medio de NSC. </li><li> El recuento celular se realizó como se describió anteriormente. </li><li> Las células se sembraron a una concentración de 5x10 4 células / ml en medio completo NSC complementado con factores de crecimiento y se colocaron en recipientes de tamaño apropiado de cultivo de tejidos como se describe en la parte anterior. </li><li> Neuroesferas secundaria se forman en 7-10 días cuando se incuban a 37 ° C en un ambiente húmedoficados incubadora con 5% de CO 2. </li></ol> 5. Los resultados representativos: Después de la siembra de las células individuales cosechados a partir de tejido del tumor GBM, tumor, como las células madre proliferan y se generan pequeños grupos de células compone de unas pocas células en 3-4 días (ver el video y la Figura 1). A medida que estos grupos crecen, adquieren una forma más esférica, para que por 7-8 días, propias esferas fase brillante, con un diámetro promedio de 150 a 200 micrones forma (Vea el video y la Figura 2). A mayor aumento, las esferas de salud por lo general demuestran microspikes en su periferia. Tener gran esfera como racimos de 1-2 días después del inicio de la cultura se debe a la existencia de grupos no disociada de la borrachera de la cultura y no debe ser confundido con las esferas verdad. La cantidad de desechos en cultivo primario esfera tumor varía en función de la fuente inicial de los tejidos y si es o no incluye cualquier tejido cerebral circundante. Técnicas adecuadas de preparación de tejidosincluyendo la disociación enzimática y mecánica, y la posterior filtración de la muestra con una cantidad suficiente de medio puede resultar en menos residuos en la cultura. <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="Figura 1" /> Figura 1. Cultivo primario esfera GBM cuatro días después de la siembra. Madre tumorales-como las células proliferan y generan pequeños grupos de células en 3-4 días. Original de ampliación; 20x. <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="Figura 2" /> Figura 2. Paso una cultura de ámbito GBM ocho días después de la siembra. Original de ampliación; 20x.

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods Mol Biol. 750, 61-77 (2011).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457-e2457 (2011).</li><li>Eramo, A. <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+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15, 504-514 (2008).</li><li>Patrawala, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy.">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl. Cancer. Inst. 100, 672-679 (2008).</li><li>Galli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</li><li>Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+label-retaining+tumour-initiating+cells+in+human+glioblastoma.">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134, 1331-1343 (2011).</li></ol>

No hay conflictos de interés declarado.

Para aislar madre neurales y células progenitoras de adulto normal y el cerebro fetal 1, 2, 3, 4 y también madre tumorales-como las células de cáncer de los tejidos como el pulmón 5, 6 de próstata, mama y cerebro 7 8 9, el ensayo ha sido neuroesfera utiliza con frecuencia como el método de elección. El uso de este ensayo simple y reproducible, se puede generar un número indefinido de las células del tejido del tumor resecado que muestran características similares a las células madre somáticas; multipotencia ex vivo, la capacidad de crear nuevos tumores tras la implantación, y auto-renovación. Estas células podrían utilizarse para estudiar la biología de las células cancerosas básicas, incluyendo las interacciones de célula a célula, y la diferenciación, la migración de la invasión, y la muerte celular. Además, tumorales aisladas como células madre constituyen una herramienta valiosa para estudiar cómo se forman los tumores, el progreso, y la recaída, y también para desentrañar los mecanismos celulares subyacentes derivados que eventualmente podría aportar información a la terapéuticaopciones.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Tipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> NeuroCult NSC medio base (Humana) </td><td> Medio </td><td> Madre Cell Technologies </td><td> 05750 </td><td></td></tr><tr><td> NeuroCult NSC proliferación Suplementos (Humana) </td><td> Suplemento de medio </td><td> Madre Cell Technologies </td><td> 05753 </td><td></td></tr><tr><td> 0,05% de tripsina-EDTA </td><td> Reactivo </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> * MEM </td><td> Reactivo </td><td> Gibco </td><td> 41500-018 </td><td> HEM componente </td></tr><tr><td> * HEPES </td><td> Reactivo </td><td> Sigma </td><td> H4034 </td><td> HEM componente </td></tr><tr><td> * Agua destilada </td><td> Reactivo </td><td&gt; Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> ** DNasa I </td><td> Reactivo </td><td> Roche </td><td> 104159 </td><td></td></tr><tr><td> ** Inhibidor de tripsina de soja </td><td> Reactivo </td><td> Sigma </td><td> T6522 </td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> Reactivo </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> N º 10 hoja de bisturí </td><td> Herramienta quirúrgica </td><td> BD </td><td> 371610 </td><td></td></tr><tr><td> Petri </td><td> Cultura software </td><td> BD Falcon </td><td> 353003 </td><td></td></tr><tr><td> Pequeñas pinzas </td><td> Los instrumentos quirúrgicos </td><td> Herramientas de Bellas Ciencia </td><td> 11050-10 </td><td></td></tr><tr><td> Células colador </td><td> Tamiz </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> T25 frasco </td><td> Cultura software </td><td> Nalge Nunc internacionales </td><td> 136196 </td><td></td></tr><tr><td> T80 frasco </td><td> Cultura software </td><td> Nalge Nunc internacional </td><td> 178905 </td><td></td></tr><tr><td> Tubos de 15 ml </td><td> Cultura software </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> Tubos de 50 ml </td><td> Cultura software </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> FCE </td><td> Factor de crecimiento </td><td> I + D </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Factor de crecimiento </td><td> I + D </td><td> 3139-FB </td><td></td></tr><tr><td> Heparina </td><td> Factor de crecimiento </td><td> Sigma </td><td> H4784 </td><td> Reconstituido en PBS </td></tr></tbody></table> * Para hacer HEM, mezcla de paquetes 1x10L del MEM and160ml de HEPES 1M y obtener un volumen de 8,75 L con agua destilada. Ajuste el pH final de 7,4 unad se almacena a 4 º C. ** Para preparar la solución de inhibidor de tripsina, en primer lugar hacer 10 ml de DNasa solución I (100 mg DNasa disueltos en 100 ml de HEM) y luego añadir 0,14 g de inhibidor de tripsina a la solución de DNasa y, finalmente, completar el volumen hasta 1 litro con HEM. Mantener las alícuotas de los productos finales en el congelador a -20 ° C.

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Como las células madre han sido aislados en los tumores, como mama, pulmón, colon, próstata y cerebro. Una cuestión fundamental en todos estos tumores, especialmente en mutliforme glioblastoma (GBM), es el de identificar y aislar células tumorales de la población de iniciar (s) para investigar su papel en la formación del tumor, la progresión y la recurrencia. Tumor comprensión de iniciar las poblaciones de células proporcionará pistas para encontrar enfoques terapéuticos efectivos para estos tumores. El ensayo neuroesfera (NSA), debido a su sencillez y reproducibilidad ha sido utilizado como el método de elección para el aislamiento y la propagación de muchas de estas células tumorales. Este protocolo demuestra el método de cultivo neuroesfera para aislar y expandir madre-como las células en el tejido resecado quirúrgicamente de tumores humanos GBM. Los procedimientos incluyen una digestión química inicial y disociación mecánica del tejido del tumor y, posteriormente, chapado a la suspensión celular resultante único de la cultura NSA. Después de 7-10 días, primaria neuroesferas de 150-200 micras de diámetro pueden ser observados y están listos para más pases y expansión.

Watch this Scientific Journal Video about Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay at JoVE.com

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Este protocolo de vídeo se muestra el aislamiento y la expansión de las células madre como de mutliforme resecado quirúrgicamente de glioblastoma humano (GBM) del tejido tumoral usando el método de ensayo de cultivo neuroesfera.

El aislamiento y la expansión de las células de glioblastoma humano multiforme tumor mediante el ensayo de neuroesfera

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

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Research

JoVE Journal

Neuroscience

29.6K vistas.  University of Florida. Este protocolo de vídeo se muestra el aislamiento y la expansión de las células madre como de mutliforme resecado quirúrgicamente de glioblastoma humano (GBM) del tejido tumoral usando el método de ensayo de cultivo neuroesfera.

Video: Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

El aislamiento y la expansión de las células troncales nerviosas de ratones adultos mediante el ensayo de neuroesfera

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Investigación

Neurociencias

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

El establecimiento de embriones de ratón con células madre neurales Cultura mediante el ensayo de neuroesfera

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Aislamiento y cultivo de células humanas las papilas fungiformes gusto

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-reprogramación de células derivadas de pericitos del cerebro humano adulto en neuronas inducidas

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

La medición cuantitativa de los niveles relativos de ácido retinoico en E8.5 embriones y Neurosphere culturas utilizando el ensayo indicador basado en células raras-lacZ F9

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Análisis de la localización de ARNS spliceosomal en células hela humanas con microinyección

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Derivación, expansión, criopreservación y caracterización de células endoteliales microvasculares cerebrales de células madre pluripotentes inducidas por humanos

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Shuttle Box Assay como herramienta de aprendizaje asociativo para la evaluación cognitiva en estudios de aprendizaje y memoria utilizando pez cebra adulto

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

Aislamiento, cultivo y caracterización de células primarias de Schwann, queratinocitos y fibroblastos del prepucio humano

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...