Este trabalho foi financiado por doações do Centro de Pesquisa Florida tumor cerebral; Preston A. Wells Jr. Centro de Terapia tumor cerebral.

1. Coleta de Tecido GBM primário <ol><li> A glioblastoma mutliforme tumor (GBM) é obtido a partir de um paciente diagnosticado com o câncer e submetidos a cirurgia. </li><li> Providências devem ser feitas com a equipe de neurocirurgia. O neurocirurgião vai colocar o tumor ressecado GBM em um tubo de tamanho apropriado que contém células-tronco neurais (NSC) meio basal suplementado com antibióticos 10-15% (Peniciline / estreptomicina). Meio frio deve ser fornecida à sala de cirurgia por laboratório. Alternativamente, frio HEPES-buffered meio mínimo essencial (HEM) ou tampão fosfato salino (PBS) com alta concentração de antibióticos também pode ser usado para esta finalidade. </li><li> O tecido do tumor ressecado é entregue ao laboratório em gelo e colocado sob o capô. </li></ol> 2. Dissociação do tumor primário em uma única célula Suspensão <ol><li> Excesso do meio original / PBS é removido do tubo falcon ea amostra é lavado 2-3 vezes com 5-10ml de PBS / NSC meio basal para remover o sangue e detritos. A PBS / Medium é removido eo tecido do tumor GBM é colocado em uma placa de Petri. </li><li> O tecido é cortado em pedaços pequenos e picada com uma lâmina de bisturi n º 10 em pedaços pequenos para aumentar a área de superfície para o processo de tripsinização. Picadura pode demorar alguns minutos 1-3, dependendo do tamanho do tumor. </li><li> O tecido é picada tripsinizados em 3-5ml de pré-aquecido 0,05% tripsina-EDTA por 10-15 minutos em banho-maria a 37 ° C. Uma pipeta eletrônico é usado para transferir as peças de tumor pequeno e tripsina em um tubo Falcon 15ml. </li><li> Um volume igual de inibidor de tripsina de soja é adicionada para parar a reacção enzimática de tripsina após o período de incubação. </li><li> Inativação da tripsina é assegurada por pipetagem de suspensão para cima e para baixo várias vezes. Então, a suspensão é peletizada para baixo por centrifugação a 800rpm (110g) para 5min. </li><li> O sobrenadante é descartado e os pedaços de tecido são suspensas em 1 ml de estéril NSC bamédio sal. Os aglomerados são dissociados por pipetagem suavemente para cima e para baixo (3-7 vezes) até que uma suspensão de células lisas única leitosa é obtida. O número de etapas de pipetagem depende diretamente do tamanho das partículas no tecido picada. Longa e vigorosa dissociação mecânica deve ser evitado, pois pode resultar em morte celular e uma redução na formação de esfera. </li><li> Para remover un-dissociada pedaços e fragmentos, 10-15 ml de meio basal é adicionado ao tubo e da suspensão de células é filtrada através de um filtro celular 40 mícrons em um tubo de 50ml. </li><li> A suspensão é filtrada centrifugado a 800rpm (110g) para 5min. O sobrenadante é descartado depois. </li><li> Pelletted células são então ressuspenso em 1-2ml de meio de NSC completa para contagem das células. </li></ol> 3. Contagem de Células e Galvanização <ol><li> 10 mL da suspensão de células é adicionado a 90 mL de 0,04% Trypan azul em um tubo eppendorf de 1ml. Nota: outras células diluição adequadações também podem ser usados. </li><li> Pipeta cima e para baixo para misturar a suspensão. 10 L das células / mistura de azul de tripano é transferido para hemocitômetro para contagem de densidade celular. </li><li> Células são banhados em meio NSC completa (uma mistura de NSC meio basal e complementar NSC proliferação em uma proporção de 9:1), suplementado com 20ng/mL EGF, bFGF e 10ng/ml 1μl/ml de heparina 0,2% (2μg/ml) em vasos de cultura apropriado tecido. 5, 20 e 40 ml de meio é usado para frascos T25, T80 e T175, respectivamente. Antibióticos podem ser adicionadas ao meio em uma concentração de 1:100 para diminuir chances de contaminação. </li><li> O balão é colocado em uma incubadora regulada a 37 ° C e 5% CO 2. </li></ol> 4. Passaging e expansão do GBM esferas derivados: <ol><li> Quando o neurospheres chegou a um tamanho médio de 150-200 m de diâmetro, a cultura está pronta para subcultura. O conteúdo de cada balão é retirado e colocado em um tamanho adequado tiss estérilue tubo de cultura, e centrifugados a 800 rpm (110 g) por 5 min à temperatura ambiente. </li><li> O sobrenadante é removido eo pellet é ressuspenso em 1 ml de 0,05% tripsina-EDTA. </li><li> Para conseguir um ótimo tripsinização, a suspensão celular é incubada a 37 ° C em banho-maria por 2-3 min. Para interromper a atividade de tripsina um volume igual de inibidor de tripsina de soja é adicionada à suspensão de células e suspensão de células é suavemente pipetado cima e para baixo. </li><li> A suspensão celular é centrifugado a 800 rpm (110g) por 5 min. Em seguida, o sobrenadante é removido e as células são ressuspendidas em 1 ml de meio de NSC. </li><li> A contagem de células é realizada conforme descrito anteriormente. </li><li> As células são banhados em uma concentração de 5x10 4 células / ml em meio NSC completo suplementado com fatores de crescimento e semeadas em vasos de tamanho apropriado de cultura de tecidos como descrito na parte anterior. </li><li> Neurospheres secundário são formados no dia 10/07 quando incubados a 37 ° C em um úmidoified incubadora com 5% de CO 2. </li></ol> 5. Resultados representativos: Após o plaqueamento das células individuais colhidos GBM tecido tumoral, tumor de células-tronco como proliferar e gerar pequenos aglomerados de células composto por poucas células em 3-4 dias (veja o vídeo e Figura 1). Como estes grupos crescem, eles adquirem uma forma mais esférica de modo que, 7-8 dias, esferas fase adequada brilhante com um diâmetro médio de 150-200 formulário microns (Veja o vídeo e Figura 2). Na maior ampliação, esferas saudável geralmente demonstram microspikes em sua periferia. Tendo como grande esfera clusters em 1-2 dias após o início da cultura é devido à existência de não-dissociada aglomerados na binging da cultura e não deve ser confundido como esferas de fato. A quantidade de detritos na cultura esfera tumor primário varia de acordo com a fonte inicial de tecido e se é ou não inclui qualquer tecido cerebral circundante. Técnicas de preparação adequada do tecidoincluindo dissociação enzimática e mecânica, e posterior filtração da amostra com uma quantidade suficiente de médio pode resultar em menos detritos na cultura. <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="Figura 1" /> Figura 1. Esfera da cultura primária GBM quatro dias após o plaqueamento. Tumor-tronco como células proliferam e geram pequenos aglomerados de células em 3-4 dias. Ampliação original; 20x. <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="Figura 2" /> Figura 2. Passage uma cultura esfera GBM 8 dias após o plaqueamento. Ampliação original; 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>

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Para isolar-tronco neurais e células progenitoras de adultos normais e cérebros fetal 1, 2, 3, 4 e também tumor-tronco como células dos tecidos do câncer, como pulmão 5, 6 de próstata, mama e cérebro 7 8, 9, o ensaio foi neurosphere freqüentemente usado como o método de escolha. Com este ensaio simples e reprodutível, pode-se gerar um número indefinido de células do tecido do tumor ressecado que mostram características similares como células-tronco somáticas; multipotency ex vivo, a capacidade de criar novos tumores após o implante, e auto-renovação. Essas células poderiam ser usadas para estudar a biologia celular básica, incluindo câncer de célula a célula-interações, e, a diferenciação de migração, invasão e morte celular. Além disso, isolado tumor-tronco como células fornecem uma ferramenta valiosa para estudar como os tumores de forma, o progresso ea recaída e também para desvendar os mecanismos celulares subjacentes decorrentes que, eventualmente, poderiam fornecer insights para terapêuticaopções.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Tipo </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> NeuroCult NSC Basal Medium (Humana) </td><td> Médio </td><td> Stem Cell Technologies </td><td> 05750 </td><td></td></tr><tr><td> NeuroCult NSC Suplementos Proliferação (Humana) </td><td> Suplemento médio </td><td> Stem Cell Technologies </td><td> 05753 </td><td></td></tr><tr><td> 0,05% de tripsina-EDTA </td><td> Reagente </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> * MEM </td><td> Reagente </td><td> Gibco </td><td> 41500-018 </td><td> Componente HEM </td></tr><tr><td> * HEPES </td><td> Reagente </td><td> Sigma </td><td> H4034 </td><td> Componente HEM </td></tr><tr><td> * A água destilada </td><td> Reagente </td><td&gt; Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> ** DNase I </td><td> Reagente </td><td> Roche </td><td> 104159 </td><td></td></tr><tr><td> ** Inibidor de tripsina de soja </td><td> Reagente </td><td> Sigma </td><td> T6522 </td><td></td></tr><tr><td> Pen / Strep </td><td> Reagente </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> No. 10 lâmina de bisturi </td><td> Ferramenta cirúrgica </td><td> BD </td><td> 371610 </td><td></td></tr><tr><td> Petri Dish </td><td> Cultura software </td><td> BD Falcon </td><td> 353003 </td><td></td></tr><tr><td> Pequeno fórceps </td><td> Instrumentos cirúrgicos </td><td> Multa Ferramentas Ciência </td><td> 11050-10 </td><td></td></tr><tr><td> Coador celular </td><td> Peneira </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> T25 frasco </td><td> Cultura software </td><td> Nalge Nunc internacionais </td><td> 136196 </td><td></td></tr><tr><td> T80 frasco </td><td> Cultura software </td><td> Nalge Nunc internacionais </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> EGF </td><td> Fator de crescimento </td><td> R &amp; D </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Fator de crescimento </td><td> R &amp; D </td><td> 3139-FB </td><td></td></tr><tr><td> Heparina </td><td> Fator de crescimento </td><td> Sigma </td><td> H4784 </td><td> Reconstituído em PBS </td></tr></tbody></table> * Para fazer HEM, mix de pacotes 1x10L de MEM and160ml de HEPES 1M e obter um volume de 8,75 L com água destilada. Definir o pH final para 7,4 umd armazenar a 4 ° C. ** Para preparar solução inibidora de tripsina, primeiro fazer 10 ml de solução de DNase I (100 mg DNase dissolvida em 100 ml de HEM) e em seguida, adicionar 0,14 g de inibidor de tripsina para a solução DNase e, finalmente, tornar o volume até 1 litro utilizando HEM. Manter as alíquotas dos produtos finais em -20 ° C freezer.

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

-Tronco como células foram isoladas em tumores como mama, pulmão, cólon, próstata e cérebro. Uma questão crítica em todos estes tumores, especialmente em mutliforme glioblastoma (GBM), é identificar e isolar a população de células tumorais iniciar (s) para investigar o seu papel na formação de tumores, progressão e recorrência. Tumor compreensão iniciar populações de células irá fornecer pistas para encontrar abordagens terapêuticas eficazes para estes tumores. O ensaio neurosphere (NSA), devido à sua simplicidade e reprodutibilidade tem sido utilizado como o método de escolha para isolamento e propagação de muitas das células tumorais isso. Este protocolo demonstra o método de cultura neurosphere para isolar e expandir-tronco como células no tecido humano ressecado cirurgicamente GBM tumor. Os procedimentos incluem uma digestão química inicial e dissociação mecânica do tecido tumoral e, posteriormente, chapeamento da suspensão de células resultantes único da cultura NSA. Após 70-10 dias, neurospheres primária de 150-200 m de diâmetro podem ser observadas e estão prontos para mais passaging e expansão.

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 vídeo demonstra o isolamento e expansão de células-tronco como de mutliforme glioblastoma humano ressecado cirurgicamente (GBM) tecido tumoral utilizando o método de cultura neurosphere ensaio.

Isolamento e Expansão da Human Glioblastoma Multiforme Células Tumorais Utilizando o Ensaio Neurosphere

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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Neuroscience

29.6K visualizações.  University of Florida. Este protocolo vídeo demonstra o isolamento e expansão de células-tronco como de mutliforme glioblastoma humano ressecado cirurgicamente (GBM) tecido tumoral utilizando o método de cultura neurosphere ensaio.

Vídeo: 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.

Isolamento e Expansão do Rato Adulto células-tronco neurais utilizando o ensaio Neurosphere

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 ...

Neurociência

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.

Estabelecer embrionárias de rato Cultura de Células-tronco neurais utilizando o ensaio Neurosphere

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.

Isolamento e Cultura de Células Papila fungiforme Humanos Taste

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-reprogramação de células pericyte derivados do Adulto Cérebro Humano em neurônios induzidos

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.

Medição quantitativa de níveis relativos ácido retinóico em E8.5 embriões e neurosphere culturas Usando a F9 RARE-Lacz baseado em células de Ensaio Reporter

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.

Análise da localização de snRNA Spliceosomal em células HeLa humanas usando microinjeção

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.

Derivação, Expansão, Criopreservação e Caracterização de Células Endoteliais Microvasculares Cerebrais de Células-Tronco Pluripotentes Induzidas Humanas

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 uma ferramenta de aprendizagem associativa para avaliação cognitiva em estudos de aprendizagem e memória usando zebrafish 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.

Isolamento, cultura e caracterização de células primárias de schwann, queratinócitos e fibroblastos de Foreskin 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.

Células Semelhantes à Microglia Humana: Diferenciação de Células-Tronco Pluripotentes Induzidas e Ensaio de Fagocitose de Células Vivas In Vitro usando Sinaptossomos Humanos

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