Este trabalho foi apoiado por Mérito comentário concessão (NEUD-004-07F) da Administração de Veteranos (para SP).

1. Expansão dos NPCs Humanos (Figura 1) <ol><li> Reviver o lote congelado de NPCs humanos a partir de cérebro fetal (Lonza, Walkersville, MD, EUA) e cultura deles em suspensão em frascos T-75 como neuroesferas (Figura 1A), em meio neurobasal contendo suplementos de proliferação, EGF (10 ng / ml) e FGF (10 ng / ml). </li><li> Após 3 dias em cultura, as neuroesferas transferir para um tubo de 15 ml e centrifugar a 500 rpm durante 5 min. </li><li> Descartar o sobrenadante deixando ~ 100 ul de gama média acima do pellet celular e transferir para um tubo Eppendorf. A pelota de 100 células mL é suficiente para dividir em dois T-75 frascos. Se menos, reduzir o número de garrafas que as células não conseguem proliferar neuroprogenitoras se dividir fina. </li><li> Tritura-se o sedimento com 200 ul de ponta 50x, mantendo a ponta 200 ul contra o fundo do tubo. Divida a suspensão de células igualmente em duas partes e adicioná-los a 2 T-75 frascos (1-2 split), um para a continuidadeexpansão e outro para a diferenciação. </li><li> Continuar a cultura das células neuroprogenitoras para expansão (primeiro balão), alterando o meio cada 4-5 dias, até que as neuroesferas atingir o seu tamanho original de 300-500 mM. Mude meio por centrifugação (500 rpm, 5 min), descarte o meio de idade e adicionar novo meio. </li></ol> 2. Diferenciação de NPCs Humano em uma Cultura Neuron-ricos (Figura 2) <ol><li> Para a diferenciação das células em cultura neuroprogenitoras rico neurónio, colocar uma única lamela para cada poço de uma placa de 24 poços e com revestimento de 100 ug / ml de poli-L-lisina através da adição de 500 uL em cada poço. Incubar os pratos durante 30 min a RT. </li><li> Aspirar o poli-L-lisina solução e lave a placa com água estéril. </li><li> Em seguida, revestir as cavidades da placa com 500 ul de 5 ug / ml de laminina rato e incubar durante 30 min. Após a incubação, lavar os poços com PBS. </li><li> 4 dias após a splitting as células (passo 1.4), transferir os pequenos neuroesferas do frasco rotulado &quot;para a diferenciação &#39;nos pratos revestidos, a uma densidade de ~ 500 neuroesferas por poço de uma placa de 24 poços. </li><li> Depois de 6 horas, quando as neuroesferas anexar ao prato, remover o meio de proliferação e diferenciação adicionar meio consistindo em meio neurobasal, suplemento B27, o NGF (20 ng / ml), BDNF (10 ng / ml), a DBC (100 mM), e ácido retinóico (2 uM). </li></ol> 3. Coloração de imunofluorescência diferenciadas NPCs (Figura 3) <ol><li> Após cultura de células neuroprogenitoras em meio a diferenciação neuronal durante duas semanas, uma cultura rica em neurônio é obtida. Lavar os neurónios uma vez com PBS e, em seguida, corrigi-los em paraformaldeído a 4% durante 30 min. Uma vez fixo, lavar as células três vezes com PBS. </li><li> Incubar as células com tampão de permeabilização (5% de BSA e 0,2% de Triton X-100 em PBS) à temperatura ambiente durante 60 min. </li><li> Incubar os pratos O / N a 4 ° C ind agitador com a seguinte combinação de anticorpos monoclonais e policlonais em 3% de BSA em PBS: NeuN (1:250) e sinapsina (1:250); acetilcolinesterase (1:500) e sinaptofisina (1:250); BDNF (1 : 500) e GAP43 (1:250); STAT3 (1:500) e GFAP (1:1.000). </li><li> Lavar as lamelas três vezes com PBS e depois incubar com anti-coelho-Cy3 e anticorpos secundários, anti-ratinho FITC no escuro à TA durante 90 min. A seguir à incubação, as lamelas lavar três vezes com PBS. </li><li> Coloque 10 ml de meio de montagem sobre uma lâmina de vidro, tirar a lamela da placa de cultura com uma pinça, e colocá-lo de cabeça para baixo no meio de montagem. Delicadamente, limpe qualquer meio de montagem excesso e selar as bordas com unha polonês. </li><li> Examine os neurônios imunomarcadas em um microscópio de fluorescência. </li></ol> 4. Captura Laser Microdissecção de neurônios (Figura 4) <ol><li> Diferenciar NPCs em uma cultura rica em neurônios em PEN slides membranas revestidos com poli-L-lisina e laminina rato seguindo os procedimentos descritos para a Figura 2. </li><li> Executar todas as etapas subseqüentes em condições RNase. </li><li> Mancha as culturas usando HistoGene Stain (Arcturus) para visualizar as células. </li><li> Encontrar as áreas de populações neuronais puros desprovidos de astrócitos, utilizando a imagem de mapa de estrada de todo o slide com o sistema Veritas LCM. Marque os neurônios usando as ferramentas de desenho. </li><li> Realizar captura a laser destas áreas utilizando precisão e automação controlado por computador num processo de dois passos. Primeiro, IR (infravermelho) é acionado em vários pontos com um ajuste de potência do laser de 70 mW e um pulso de 2.500 ms para obter a membrana anexar ao cap. Em seguida, a área marcada é extirpado usando uma ferramenta de corte UV para um nível baixo nível de 10 mV. </li><li> Combinação destes processos de captura selectivamente as zonas marcadas a partir da membrana para PEN CapSure LCM macro cápsulas. Quando a tampa é levantada, a membrana com o neurons adere à tampa. </li><li> Isolar o RNA total a partir de amostras de LCM, utilizando um kit de isolamento de RNA PicoPure (Arcturus) e trata-se com ADNase. </li><li> Amplificar o ARN isolado utilizando o kit de amplificação de RNA RiboAMP seguindo as instruções do kit. </li><li> Realizar a análise em tempo real de RT-PCR utilizando sondas TaqMan para detectar neurofilamento humano de cadeia pesada (hNFHc). </li></ol> Abreviações: AD, doença de Alzheimer, o BDNF, o factor neurotrófico derivado do cérebro, CREB, cíclico proteína de ligação de elemento de resposta a AMP; DBC, dibutiril AMP cíclico; EGF, factor de crescimento epidérmico; FGF, factor de crescimento de fibroblastos; GFAP, proteína ácida fibrilar glial, LCM, laser capturar microdissection; NGF, fator de crescimento do nervo; NPC, neuroprogenitoras célula; PEN, naftalato de polietileno.

<ol>
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Não há conflitos de interesse a declarar.

Nós descrevemos neste estudo um modelo de cultura de células ricas em neurônio pela diferenciação de células neuroprogenitoras humanos auto-renovação e um método para isolar uma população pura de neurônios por captura de microdissecção a laser. Nós usamos uma combinação de NGF, BDNF, DBC e ácido retinóico para a diferenciação neuronal de NPCs. DBC é usada para ativar CREB, um fator de transcrição que aumenta a neurogênese 12. O ácido retinóico induz saída do ciclo celular e reduz a...

Neurogénese longo da vida, é conhecido por ocorrer na zona subventricular dos ventrículos laterais e na camada subgranular do giro dentado do cérebro de mamíferos adultos 1. Células neuroprogenitoras (NPCs) que se originam a partir destas regiões são células multipotentes que podem se diferenciar em neurônios, astrócitos e oligodendrócitos 2. NPCs gerado interesse devido ao seu potencial para ser transplantadas em pacientes com várias doenças neurodegenerativas, incluindo a doença de Parkinson, esclerose lateral amiotrófica, acidente vascular cerebral e doença de Alzheimer (AD) 3. Estudos com NPCs geralmente centrada sobre este ângulo de transplante, mas o potencial de neurónios NPC-derivados como um modelo de cultura de células para determinar o mecanismo de neurodegeneração não foi totalmente explorada. Estudos anteriores têm geralmente utilizados os neurónios pós-mitóticos, isolado a partir de tecidos do cérebro de roedores, que devem ser isoladas para cada experiência em que não sejamauto-renovação. Embora as linhas celulares de neuroblastoma humano, incluindo SH-SY5Y e células SK-N-MC pode ser expandido, elas não têm as características de neurónios primários. NPCs humanos, por outro lado, oferecem vantagens porque eles podem ser expandidos para várias passagens e podem ser diferenciados para gerar uma população de células com as características primárias de neurónios 4,5. No presente estudo, descrevemos um novo protocolo de diferenciação para se obter uma população rica em neurônio consistente de NPCs disponíveis comercialmente isoladas de cérebro fetal humano. Porque essas culturas contêm uma pequena porcentagem de células gliais, precisamos de métodos adicionais para isolar uma população pura de neurônios para caracterização molecular. Captura laser microdissecção (LCM) é um nova técnica pela qual uma população homogénea de células a partir de uma secção de tecido podem ser capturadas selectivamente para análise da expressão do gene 6. O cérebro é um tecido heterogêneo composto por neurônios, células gliais e outros ty celularpes. LCM foi utilizado para determinar a expressão de genes específicos de neurónios analisa 7-10. Temos realizado anteriormente LCM de neurônios do hipocampo de AD (Tg2576) seções do cérebro do rato para mostrar a diminuição da expressão de proteína de ligação elemento de resposta ao AMP cíclico (CREB) e BDNF especificamente nos neurônios do hipocampo 11. No presente estudo, descrevemos os procedimentos para a expansão dos NPCs humanos, a diferenciação neuronal, coloração de imunofluorescência para marcadores neuronais e LCM para o isolamento de neurônios cultivados em PEN lâminas de membrana.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Neuroprogenitor cells (NPCs) </td>
			<td>Lonza</td>
			<td>PT-2599</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A human basal medium</td>
			<td>Stem cell technology</td>
			<td>5750</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Proliferation supplement</td>
			<td>Stem cell technology</td>
			<td>5753</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Differentiation supplement</td>
			<td>Stem cell technology</td>
			<td>5754</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B-27 Supplement (50x) </td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human epidermal growth factor</td>
			<td>Stem cell technology</td>
			<td>2633</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fibroblast growth factor </td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Brain Derived Neurotrophic Factor </td>
			<td>Cell signaling </td>
			<td>3897S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nerve Growth Factor </td>
			<td>Invitrogen</td>
			<td>13257-019</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibutyryl cyclic AMP</td>
			<td>Sigma</td>
			<td>D-0627</td>
			<td></td>
		 </tr>
		 <tr>
			<td>poly-L-lysine </td>
			<td>Sigma</td>
			<td>P-5899</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse laminin </td>
			<td>Sigma</td>
			<td>L-2020</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R-2625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>STAT3 antibody</td>
			<td>Cell signaling</td>
			<td>9132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFAP antibody</td>
			<td>Cell signaling</td>
			<td>3670</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GAP43 antibody</td>
			<td>Transduction laboratories</td>
			<td>612262</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BDNF antibody</td>
			<td>Millipore</td>
			<td>AB1534SP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetyl cholinesterase antibody </td>
			<td>Santz cruz</td>
			<td>Sc-11409</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synaptophysin antibody</td>
			<td>Abcam</td>
			<td>ab18008-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NeuN antibody</td>
			<td>Chemicon</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synapsin antibody</td>
			<td>Novus</td>
			<td>NB300-104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse FITC</td>
			<td>Jackson Immuno </td>
			<td>115-095-146</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti Rabbit Cy3</td>
			<td>Jackson Immuno </td>
			<td>711-165-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triton-X 100</td>
			<td>Acros</td>
			<td>21568-2500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Paraformaldehye </td>
			<td>Fisher</td>
			<td>4042</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip (Big circle cover slip)</td>
			<td>Fisherbrand</td>
			<td>12-545-102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mounting medium (Prolong Gold)</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen membrane</td>
			<td>Applied biosystems</td>
			<td>LCM0521</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Histogene LCM frozen section staining kit</td>
			<td>Applied biosystems</td>
			<td>KIT0401</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboAmp RNA Amplification kit</td>
			<td>Applied biosystems</td>
			<td>KIT0201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Picopure RNA Isolation kit</td>
			<td>Applied biosystems</td>
			<td>KIT0202</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CapsureMacro LCM caps</td>
			<td>Applied biosystems</td>
			<td>LCM0211</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture

Células neuroprogenitoras (NPCs) isoladas a partir do cérebro de feto humano foram expandidas sob condições proliferativas em presença do factor de crescimento epidérmico (EGF) e factor de crescimento de fibroblastos (FGF), para proporcionar um fornecimento abundante de células. NPCs foram diferenciadas na presença de uma nova combinação de factor de crescimento do nervo (NGF), factor neurotrófico derivado do cérebro (BDNF), dibutiril AMPc (DBC) e ácido retinóico em pratos revestidos com poli-L-lisina e laminina rato para obter neurónio ricas culturas. NPCs também foram diferenciadas na ausência de neurotrofinas, a DBC e ácido retinóico e na presença do factor neurotrófico ciliar (CNTF) para produzir culturas ricas em astrócitos. NPCs diferenciadas foram caracterizadas por coloração de imunofluorescência para um painel de marcadores neuronais incluindo NeuN, sinapsina, acetilcolinesterase, sinaptofisina e GAP43. Proteína glial fibrilar ácida (GFAP) e STAT3, marcadores de astrócitos, foram detectados em 10-15% dos NPCs diferenciadas. Para facilitarCaracterização molecular específicos do tipo de célula, a captura de microdissecção a laser foi realizada para isolar neurónios cultivados em naftalato de polietileno (PEN) de lâminas de membrana. Os métodos descritos neste estudo fornecem ferramentas valiosas para avançar nossa compreensão do mecanismo molecular da neurodegeneração.

Expansão dos NPCs (Figura 1) Quando neuroesferas são quebradas até uma suspensão de células individuais por meio de trituração, a ponta da pipeta deve tocar no fundo do tubo, de modo que existe uma certa resistência, quando a suspensão é pipetado para cima e para baixo. O número de vezes de trituração vai variar entre os indivíduos e tem de ser decidido, por tentativa e erro, examinando a suspensão de células resultantes com um microscópio. É fundamental para ...

Watch this Scientific Journal Video about Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture at JoVE.com

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Células neuroprogenitoras Humanos (NPCs) foram expandidas em condições de proliferação. NPCs foram diferenciadas em culturas ricas em neurônios na presença de uma combinação de neurotrofinas. Marcadores neuronais foram detectadas pela técnica de imunofluorescência. Para isolar uma população pura de neurônios, NPCs foram diferenciadas em PEN lâminas de membrana e captura de microdissecção a laser foi realizada.

Captura Laser Microdissecção de neurônios a partir de células diferenciadas neuroprogenitoras humanas em cultura

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

laser-capture-microdissection-neurons-from-differentiated-human

Research

JoVE Journal

Neuroscience

12.1K visualizações.  Denver VA Medical Center.  Células neuroprogenitoras Humanos (NPCs) foram expandidas em condições de proliferação. NPCs foram diferenciadas em culturas ricas em neurônios na presença de uma combinação de neurotrofinas. Marcadores neuronais foram detectadas pela técnica de imunofluorescência. Para isolar uma população pura de neurônios, NPCs foram diferenciadas em PEN lâminas de membrana e captura de microdissecção a laser foi realizada.

Vídeo: Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Neurociência

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Laser Microdissecção Captura de Drosophila Neurônios periféricos

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivação eficiente dos recursos humanos Progenitores Neuronal e Neurônios de pluripotentes células estaminais embrionárias humanas com a indução de moléculas pequenas

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Laser Microdissecção Captura de Populações enriquecida de neurônios ou neurônios individuais para análise de expressão gênica após a lesão cerebral traumática

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Produção de RNA para análise transcriômica de corpos celulares de neurônio motor da medula espinhal do rato por microdisseção de captura a laser

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

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 ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Captura Laser Microdissection - uma demonstração do Isolamento de Pessoa dopamina Neurônios e região ventral tegmental Entire

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal Root Ganglia neurônios e células-tronco diferenciadas adiposo-derivados: An<em&gt; In Vitro</em&gt; Co-cultura modelo para estudar a regeneração do nervo periférico

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Estereotáxica guiada por Atlas Laser Microdissection de captura de regiões do cérebro afetadas por lesão traumática

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

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

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

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

Simples geração de uma cultura de alto rendimento de neurônios induzidas de fibroblastos de pele humana adulta

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

Methods Article

Captura Laser Microdissecção de neurônios a partir de células diferenciadas neuroprogenitoras humanas em cultura