Este trabalho foi apoiado financeiramente pela ANR (Agence Nationale de la Recherche), AFM (Association Française contre les Miopatias), FEDER em PACA e Irme (Institut de Recherche sur la Moelle épinière et l&#39;Encephale). Nós agradecem Marie Pierre Blanchard (Jean Roche Institute) por sua ajuda eficiente durante a gravação de lapso de tempo.

1. Coleção de Mucosa olfatória em Ratos <ol><li> Comece preparando três pratos de 35 milímetros Petri cheia de DMEM / HAM F12 meio de cultura em uma capa cultura limpa. </li><li> Qualquer método de eutanásia deve ser previamente aprovado pelo cuidado da instituição animal e comissão de uso e realizado por pessoal qualificado. Depois que o rato entrou anestesia profunda com pentobarbital sódico ou outras formas de anestesia injetável, tais como a cetamina / xilazina, decapitar e remover a pele. Os anestésicos inalantes deve ser evitado. Adequação da anestesia será avaliada por pinch pés antes de decapitação. Remova o maxilar inferior com uma tesoura e com a ajuda de um rongeur, eliminar os músculos faciais de ambos os lados. </li><li> A partir da volta dos incisivos, retire com uma rongeur o osso que cobre a cavidade nasal, um lado de cada vez. Cornetos olfativa entram em vista como laranja / marrom órgãos localizados na parte de trás do nariz. </li><li> Delicadamente descartar as conchas com uma pinça. Usando uma agulha de calibre 26, isolar a mucosa olfativa deitado no septo, cortando o tecido ao longo de três linhas: o arco da placa perpendicular, lâmina crivosa e teto da cavidade nasal. </li><li> Coletar biópsias de ambos os lados e transferi-los em um DMEM / HAM placa de Petri F12-cheia. Este procedimento não deve demorar mais do que 10 minutos do início da eutanásia. </li><li> Agora, a fim de remover o muco, a transferência das biópsias duas vezes em pratos de Petri cheia médio. </li></ol> 2. Coleção de Mucosa olfatória em seres humanos <ol><li> Este procedimento deve ser realizado por um Ouvido, Nariz e Garganta (ENT) cirurgião, de acordo com o comitê de ética local relevante (s), e cada ambulatorial deve assinar um termo de consentimento informado. </li><li> Usando um 0 ° ou 30 ° endoscópio rígido (4 mm de diâmetro), inspecionar ambas as fossas nasais e avaliar a presença de pólipos putativa ou qualquer lesão inflamatória. Escolher a melhor cavidade nasal, tendo em conta o desvio do septo. </li><li> Usando um aplicador de algodão, aplicar uma anestesia local, como a lidocaína com epinefrina, por 10 minutos. </li><li> Com uma pinça throughcut etmóide, coletar uma biópsia dois quadrados milímetros ou na raiz do aspecto medial da concha média ou no septo na área dorsomedial. </li><li> A biópsia olfativa é então transferido, usando uma agulha estéril, em um tubo estéril 2 ml preenchida com 1 ml de DMEM / HAM F12. Dica do tubo de cabeça para baixo para se certificar de que a biópsia está imerso em meio de cultura. </li><li> Insira o tubo em um recipiente refrigerado e transportá-lo para o laboratório de pesquisa. Nesta fase, a biópsia pode ser usado por si para estudos comparativos molecular focada em doenças específicas do cérebro ou processados ​​para gerar células-tronco. </li></ol> 3. Isolamento de células estaminais da mucosa olfativa Humanos e Rat <ol><li> Lavar as biópsias em DMEM / HAM F12. Incubar as biópsias em um prato de Petri preenchida com 1 ml de solução dispase II (2,4 UI / ml), por 1 hora a 37 ° C. </li><li> Em seguida, sob um microscópio de dissecação com uma luz difratada invertido, o epitélio olfativo é removido da lâmina própria subjacente usando uma espátula de micro. </li><li> O epitélio olfativo é mais fino e olha translúcido sobre um fundo preto em comparação com a lâmina própria que é listrada de laranja / marrom. Sobre um fundo branco, o epitélio parece cinza e lâmina própria, marrom. </li><li> Uma vez purificado, transferir a lâmina em uma placa de Petri cheia de DMEM / HAM F12. </li><li> Se o tecido é de um roedor, em seguida, corte da lâmina própria em pequenos pedaços com duas agulhas 25. Em seguida, transferir as peças para um tubo de 15 ml preenchida com 1 ml de colagenase IA. </li><li> No tubo, com uma pipeta de plástico estéril, dissociar o tecido. Então, incubar o tubo por 10 minutos a 37 ° C. </li><li> Para terminar a dissociação, agite levemente o tubo e adicionar 9 ml de Ca-Mg-livre e livre PBS e centrifugar a 200 g por 5 minutos. </li><li> Ressuspender o pellet celular em DMEM / HAM F12 meio de cultura suplementado com soro bovino fetal a 10%, antibióticos e placa em placas de cultura de plástico. </li><li> Agora, se o tecido é humano, então a fatia de lâmina em 3 a 4 peças com uma espessura variando de 200 a 500 mm. </li><li> Inserir cada tira em seu prato 2 centímetros de diâmetro própria cultura e cobrir o tecido com 1,3 centímetros de diâmetro estéril lamínulas de vidro. </li><li> Então, adicionar 500 mL de meio de cultura (DMEM / HAM F12 suplementado com soro bovino fetal 10% e antibióticos) para cada placa de cultura. </li><li> Para qualquer tipo de tecido, renovar o meio de cultura a cada 2 a 3 dias. </li><li> Cinco a sete dias após, as células-tronco começará a invadir a placa de cultura e após duas semanas devem ser confluentes. Quando confluência é atingido, a passagem e transferência das células para frascos de cultura. </li></ol> 4. Formação esfera e difere Neuronalntiation de Células-Tronco Olfactory <ol><li> Para gerar esferas de células-tronco, incubar os frascos por duas horas a 37 ° C com poli-L-lisina. (TEXTO: 5 g/cm2). </li><li> Placa as células com uma densidade de 16.000 células por centímetro quadrado nos frascos tratada. </li><li> A cada dois dias, alimentar as células com 0,2 ml por centímetro quadrado de meio suplementado (DMEM / HAM F12 suplementado com insulina, transferrina, selênio (ITS-X, 1%), EGF (50 ng / ml) e FGF2 (50 ng / ml)). </li><li> Dois a cinco dias depois, recolher as esferas de células de flutuação e ou re-plate ou dissociá-los antes do miocárdio em modelos animais de terapia celular. </li><li> Para diferenciar as células-tronco em neurônios olfativos células semelhantes, cultivá-las por 21 dias em meio contendo Neurobasal B-27, a penicilina, estreptomicina glutamina e glutamato. </li><li> Em seguida, fazer mudanças de médio a cada 3 dias. Neurônio-como pilhas deve aparecer após 2-3 semanas. </li></ol> 5. Resultados representativos: Nasal humana explante-outgrowing células-tronco (Figura 2A) estão se dividindo rapidamente e confluência pode ser alcançado dentro de 1-2 semanas. Uma característica chave da stemness, expressão nestina, foi avaliado (Figura 2B). Quando cultivada em poli-L-lisina com um meio de cultura sem soro suplementado com EGF (50 ng / ml) e FGF2 (50 ng / ml), as células-tronco olfativas dar origem a esferas (figura 2C). Quando cultivada em soro contendo meio de cultura recém-esferas banhado dar origem a células que expressam GFAP (~ 50%), tubulina células que expressam (~ 10-15%) e O4 células que expressam (~ 2-5%) 9 (Figuras 2D-F). No entanto, o destino das células-esfera derivada pode ser modificado. Por exemplo, quando cultivada em meio de cultura suplementado com B27 Neurobasal e glutamato, a maioria dos nasal células-tronco em neurônios olfativos semelhantes células expressando β-tubulina III (Figura 2G) e MAP2 (Figura 2H). <img src="/files/ftp_upload/2762/2762fig1.jpg" alt="Figura 1"/> Figura 1. Esquema geral do experimento. Biópsias mucosa olfativa são excisadas de rato ou cavidade nasal humana. Explantes podem ser usados ​​por si só para fins comparativos, estudos moleculares visando identificar biomarcadores em doenças cérebro. Para isolar células-tronco olfativas, as interações entre a lâmina própria eo epitélio neuro-são interrompidas com o II dispase enzima e, após 45 minutos, o epitélio é removido com uma espátula micro. Células-tronco de roedores olfativos são mais selecionados por dissociar a lâmina com colagenase IA. Para o tecido humano, pedaços de lâmina olfativa são cultivadas em lamínula de vidro até que as células-tronco outgrowing invadir todo o bem. Depois de proliferação, usando um meio de cultura apropriado, células-tronco olfativas pode gerar esferas ou se diferenciar em neurônio-como pilhas. Células-tronco olfativas pode ser usado para i) doenças reparação cerebral ou trauma ou ii) identificar marcadores moleculares do centro de doenças do sistema nervoso. Ilustrações são feitas com a ajuda de Servier Arte Médica. <img src="/files/ftp_upload/2762/2762fig2.jpg" alt="Figura 2"/> Figura 2. Cultura e diferenciação de células-tronco humanas nasal olfativo. Células-tronco humanas crescem fora da lâmina própria explante (A) se dividem rapidamente, quando cultivadas em meio de soro contendo. Células-tronco expressar a stemness marcador nestina (B). Quando semeadas em poli-L-lisina revestido de plástico e cultivadas em meio de cultura sem soro suplementado com EGF e FGF2, células-tronco olfativas gerar esferas (C). Esfera células derivadas, quando banhado no soro contendo meio de cultura, dar origem a células que expressam GFAP (~ 50%), tubulina células que expressam (~ 10-15%) e O4 células que expressam (~ 2-5%) 9 (DF). Quando cultivada em meio de cultura suplementado com B27 Neurobasal e glutamato, que se diferenciar em neurônio-como células que expressam β-tubulina III (G) e MAP2 (H).

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Não há conflitos de interesse declarados.

As técnicas apresentadas aqui fazer o roedor e mucosa olfativa humana um modelo útil para a investigação clínica sobre as causas de doenças neurodegenerativas e do desenvolvimento neurológico, bem como uma ferramenta para reparar o cérebro patológicos ou traumatizados. O protocolo é relativamente simples e pode ser facilmente realizado por um biólogo celular experientes. A taxa de sucesso para as técnicas de biópsia e cultura é alto. Etapas críticas <ul><li> Para a coleta de mucosa olfativa em roedores, é recomendado não exceder um limite de tempo de 10 minutos entre a eutanásia ea excisão final do tecido olfativo. </li><li> A dissociação da lâmina própria de roedores olfativa é geralmente obtida após 10 min. incubação em colagenase IA. Se não, recomendamos a recolher no dia seguinte o sobrenadante em que os bits não dissociado do flutuador lâmina própria, centrífuga que em 200 g e mecanicamente dissociar a lâmina flutuante usando uma pipeta Pasteur antes repique as células em um novo poço. O primeiro poço que contém as células já anexado é preenchido com meio de cultura fresco. </li><li> Para a lâmina própria humana, que é mais compacto que o tecido de roedores, não recomendamos uma dissociação enzimática. O tecido é cortado e cada explante é inserido entre o fundo do prato de plástico e uma lamela de vidro. Culturas bem sucedidas incluem explantes cuja espessura variam de 200 a 500 mm. </li></ul> Possíveis modificações <ul><li> O protocolo atual pode ser ligeiramente modificada, a fim de gerar neurônios olfativos in vitro. Para esse efeito, o neuro-epitélio não é removido ea mucosa olfativa é todo cortado com um helicóptero McIlwain (200 espessura mm). Cada explante é banhado em um prato, parcialmente secos durante uma hora e depois reidratados com FCS contendo meio de cultura. Durante os primeiros dias pós-revestimento, células epiteliais e mesenquimais crescer fora do explante. Então, progenitores neurônio vai migrar no topo desta camada de células e se diferenciar em neurônios. </li><li> Purificação de células-tronco olfativas pode ser conseguido usando citometria de fluxo. Marcadores de superfície específicos podem ser recuperadas a partir da lista publicada no jornal caracterização 11. </li><li> Para experimentos de transplante, é possível usar uma linhagem de ratos cujas olfativa células-tronco são GFP-positivo. Esta cepa de ratos (Sprague Dawley, eGFP conduzido pelo promotor PGK) está disponível em ITERT (Nantes, França). Para inserir o gene GFP em células-tronco olfativas, nós usamos um método baseado na infecção lentivírus. </li><li> Este trabalho está focado em células olfativas ecto-mesenquimais. No entanto, outro tipo de célula de interesse, as células olfativas ensheathing, pode ser purificado a partir do mesmo tecido. Nós descrevemos um método para a sua recolha e tratamento 1,17 e usamos nasal humana células ensheathing para um ensaio clínico de fase I / IIa clínicos em pacientes paraplégicos 18. </li><li> Como um membro da superfamília de células-tronco mesenquimais, OE-CTMs são capazes, sob condições de cultivo adequado, para se diferenciar em adipócitos, osteócitos e miócitos 11. </li></ul> Aplicações futuras <ul><li> Nós já usamos humana biópsias olfativa para estudar anomalias celulares e moleculares em pacientes com autismo, transtorno bipolar, disautonomia familial, doença de Parkinson, doença de Alzheimer e esquizofrenia 2-7. Teoricamente, todas as doenças do cérebro podem ser estudadas usando biópsias nasais ou periférico de células-tronco olfativas. </li><li> Roedores e humanos nasal células-tronco olfativas já foram enxertados em modelos animais de amnésia, doença de Parkinson, lesão coclear e traumas da medula espinhal 12-16. É concebível para o transplante destas células em modelos animais da doença de Alzheimer, isquemia cerebral, esclerose múltipla. </li></ul>

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td colspan="3"> Coleção de mucosa olfativa em ratos </td></tr><tr><td> DMEM / HAM F12 </td><td> Invitrogen </td><td> 31331-028 </td></tr><tr><td> Pentobarbital sódico </td><td></td><td></td></tr><tr><td> Rongeur </td><td> FST </td><td></td></tr><tr><td> Agulha de calibre 26 </td><td> Terumo </td><td> NN-2613R </td></tr><tr><td> Fórceps </td><td></td><td></td></tr><tr><td colspan="3"> Coleção de mucosa olfactiva em humanos </td></tr><tr><td> Endoscópio rígido </td><td> Karl Storz ou Richard Wolf Medical </td><td></td></tr><tr><td> Lidocaína </td><td></td><td></td></tr><tr><td> Epinefrina </td><td></td><td></td></tr><tr><td> Throughcut etmóide forceps </td><td> Karl Storz ou Richard Wolf Medical </td><td></td></tr><tr><td colspan="3"> Isolamento de células-tronco olfativas </td></tr><tr><td> Dispase II </td><td> Roche </td><td> 10 295 825 001 </td></tr><tr><td> Microscópio de dissecação </td><td></td><td></td></tr><tr><td> Espátula Micro </td><td> FST </td><td></td></tr><tr><td> Colagenase IA </td><td> Sigma-Aldrich </td><td> C9891 </td></tr><tr><td> Ca-free/Mg-free PBS </td><td> Invitrogen </td><td> 14190-250 </td></tr><tr><td> Soro fetal bovino </td><td> Invitrogen </td><td> 10270098 </td></tr><tr><td> Lamínula de vidro </td><td> Knittel Glaser </td><td> 001/35 </td></tr><tr><td colspan="3"> Formação de esfera e diferenciação neuronal </td></tr><tr><td> Poli-L-lisina </td><td> Sigma-Aldrich </td><td> P-1274 </td></tr><tr><td> Insulina transferrina selênio (ITS) </td><td> Invitrogen </td><td> 51500056 </td></tr><tr><td> EGF </td><td> R &amp; D Systems </td><td> 236-EG </td></tr><tr><td> FGF2 </td><td> R &amp; D Systems </td><td> 233-FB </td></tr><tr><td> Médio Neurobasal </td><td> Invitrogen </td><td> 21103-049 </td></tr><tr><td> B-27 Suplemento Serum-Free </td><td> Invitrogen </td><td> 17504-044 </td></tr><tr><td> Penicilina / estreptomicina </td><td> Invitrogen </td><td> 15140122 </td></tr><tr><td> Glutamina </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Glutamato </td><td> Sigma-Aldrich </td><td></td></tr></tbody></table>

isolating nasal olfactory stem cells from rodents or humans

A mucosa olfativa, situado na cavidade nasal, é responsável pela detecção de odores. É também o único tecido nervoso que está exposta ao ambiente externo e de fácil acesso em cada indivíduo vivo. Como resultado, este tecido é única para qualquer pessoa com o objetivo de identificar anomalias molecular no cérebro patológico ou isolar células-tronco adultas para terapia celular. Anormalidades moleculares em doenças cerebrais são frequentemente estudadas usando amostras de tecido nervoso coletado post-mortem. No entanto, este material tem limitações numerosas. Em contraste, a mucosa olfativa é facilmente acessível e pode ser biopsiados com segurança, sem qualquer perda de sentido do olfacto 1. Assim, a mucosa olfativa fornece uma &quot;janela aberta&quot; no ser humano adulto através da qual se pode estudar de desenvolvimento (por exemplo, autismo, esquizofrenia) 2-4 ou neurodegenerativas (Parkinson, por exemplo, Alzheimer) doenças 4,5. Mucosa olfativa pode ser usado tanto para estudos comparativos molecular 4,6 ou em experimentos in vitro na neurogênese 3,7. O epitélio olfativo também é um tecido nervoso que produz novos neurônios a cada dia para substituir aqueles que estão danificados pela poluição, bacteriana de infecções virais. Este neurogênese permanente é sustentada pelos progenitores, mas também células-tronco que residem em ambos os compartimentos da mucosa, ou seja, o neuroepitélio e 10/08 lâmina própria subjacente. Recentemente, desenvolveu um método para purificar as células-tronco adultas localizadas na lâmina própria e, depois de ter demonstrado que elas estão intimamente relacionadas com a medula óssea células-tronco mesenquimais (MSC-BM), que nomeou células olfativas ecto-mesenquimais (OE- MSC) 11. Curiosamente, quando comparado ao BM-MSCs, OE-MSCs exibir uma alta taxa de proliferação, uma clonogenicity elevada e uma inclinação de se diferenciar em células neurais. Aproveitamos essas características para realizar estudos dedicados a desvendar genes novo candidato na esquizofrenia e 4 da doença de Parkinson. Nós e os outros também têm demonstrado que OE-MSCs são promissores candidatos para terapia celular, depois de um trauma na medula espinhal 12,13, a lesão coclear 14 ou em um modelo animal da doença de Parkinson 15 ou amnésia 16. Neste estudo, apresentamos métodos para biópsia da mucosa olfativa em ratos e seres humanos. Após a coleta, a lâmina própria é enzimaticamente separada do epitélio e as células-tronco são purificados usando um enzimáticos ou um método não-enzimática. Purificada de células-tronco olfativas pode então ser cultivado em grande número e depositado em nitrogênio líquido ou induzido a formar esferas ou diferenciados em células neurais. Essas células-tronco também podem ser usados ​​para omics comparativa (genômica, transcriptomic, epigenômico, proteômica) estudos.

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

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Descrevemos aqui um método para biopsying mucosa olfativa de ratos e humanos cavidades nasais. Essas biópsias podem ser utilizados tanto para identificar anomalias molecular em doenças cerebrais ou isolar células-tronco adultas multipotentes que podem ser utilizados para o transplante de células em modelos animais de trauma cerebral / doença.

Isolando Nasal Células-Tronco olfativo dos roedores ou humanos

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

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

Research

JoVE Journal

Neuroscience

30.5K visualizações.  Aix Marseille University. Descrevemos aqui um método para biopsying mucosa olfativa de ratos e humanos cavidades nasais. Essas biópsias podem ser utilizados tanto para identificar anomalias molecular em doenças cerebrais ou isolar células-tronco adultas multipotentes que podem ser utilizados para o transplante de células em modelos animais de trauma cerebral / doença.

Vídeo: Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Electrophysiological Measurements from a Moth Olfactory System

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

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

Medidas eletrofisiológicas partir de um sistema olfativo Traça

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

Neurociência

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

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

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

Isolando-LacZ expressar Células de tecidos de camundongos Inner Ear usando Citometria de Fluxo

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

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

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

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

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

Expansão da embrionárias e adultas células-tronco neurais por<em&gt; In Utero</em&gt; Eletroporação ou injeção estereotáxica Viral

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

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

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

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

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

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.

Um Método de Expansão cGMP-aplicável para Agregados de Neural Humano-tronco e células progenitoras derivadas de células-tronco pluripotentes ou tecido cerebral do feto

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.

Derivação sem alimentador de células da crista neural progenitoras pluripotentes humanas de células-tronco

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

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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

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

Dirigido neurônios dopaminérgicos Diferenciação de Células-Tronco pluripotentes humanas

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

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

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

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

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

Modelagem astrocitoma Patogênese<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Usando Cortical astrócitos ou células-tronco neurais de condicional, Geneticamente Modificadas Ratos

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

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

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

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

Neurônios olfativos obtida através Nasal Biópsia Combinada com Laser-Capture Microdissection: Uma Abordagem Potencial para tratamento estudo Response em Transtornos Mentais

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

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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

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

Diferenciação neuronal de células-tronco embrionárias do rato in vitro

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