Name: the specification of telencephalic glutamatergic neurons from human pluripotent stem cells
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Description: Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

Os autores gostariam de agradecer ao Dr. Y. Sasai para generosamente fornecer o anticorpo FOXG1. Este trabalho foi financiado por Connecticut Stem Cell Research Grants (08-SCB-UCHC- 022 e 11-SCB24) e Fundação paraplegia espástica.

1. Geração de pluripotentes humanas Agregados de Células Tronco (D1-D4) <ol><li> As células estaminais pluripotentes humanas são cultivadas em fibroblastos embrionários de ratinho (MEF) alimentadores na presença de meio de hESC suplementado com factor de crescimento de fibroblastos básico (bFGF, 4 ng / ml). Após 5-7 dias de cultura, quando as colónias são grandes, mas ainda não-diferenciada, eles estão prontos para o próximo passo. </li><li> A solução de enzima deve primeiro ser preparado. Num tubo de ...

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Existem vários passos cruciais durante o processo de diferenciação neuronal. É importante assegurar que as PSCs são pluripotentes humanas porque de outro modo as células podem já ser inclinado para tornar-se uma linhagem não-neuronal. Isto pode ser confirmado por coloração das PSCs humanos com anticorpos contra marcadores de pluripotência como Oct4, Sox2, Nanog e Tra-1-60 1-3. Se as PSCs humanos não dão muito bem após passaging deles, ROCK inibidor (Y27632) pode ser adicionado para ajudar. Para ...

As células-tronco pluripotentes humanas (PSCs), incluindo tanto células estaminais embrionárias humanas (hESCs) e as células-tronco pluripotentes induzidas (iPSCs), têm a capacidade de gerar qualquer tipo de célula no corpo, incluindo neurónios 1-3. Diferenciação dirigida de vários subtipos neuronais de PSCs humanos é a chave para a aplicação dessas células na medicina regenerativa. A geração de subtipos funcionais neuronais durante o desenvolvimento é um processo complexo, que envolve a indução da linhagem neuronal, a especificação de progenitores regionais ao longo do eixo rostro-caudal, e a diferenciação dos tipos de pós-mitóticos neuronais a partir dos progenitores regionais 4,5. A partir de 2001, vários sistemas foram criados para gerar linhagem neural de hESCs, que forneceram uma plataforma para a geração subseqüente de subtipos neuronais 6,7. Com base em princípios de desenvolvimento, vários tipos de neurônios, como espinhal neurônios motores 8-12, mesencéfalo dopneurônios aminérgicos 13-15, e as células da retina neural 16,17 foram especificadas de forma eficiente PSCs humanos. Isto foi feito pela aplicação de morfogénios críticos que são importantes para a especificação de um destes tipos de neurónios durante o desenvolvimento in vivo. Outros protocolos também foram desenvolvidos para promover a diferenciação de neurónios em hESCs utilizando quer outros factores 18-20, tais como as moléculas pequenas, ou por co-cultura com outros tipos de células para ajudar a promover a diferenciação 21. O neocórtex humano é altamente desenvolvida e contém muitos tipos de células, incluindo neurônios glutamatérgicos que desempenham um papel importante na aprendizagem, memória e função cognitiva 22,23. O primeiro passo na geração de neurónios glutamatérgicos na cultura é especificar células progenitoras telencefálicos. Grupo Yoshiki Sasai o primeiro relataram a diferenciação de precursores dirigido telencefálicos de rato CES (mESCs) utilizando um suspensio isento de soron cultura na presença de DKK1 (que inibe a via de sinalização Wnt), bem como LeftyA (que inibe a sinalização nodal) 24. Posteriormente, vários grupos, incluindo a nossa também relataram a especificação dos precursores telencefálicos de PSCs humanos em soro meio livre 25-27. A geração de precursores de PSCs telencefálicos humanos não requer a utilização de morfogénios exógenas e a eficiência na geração destes precursores é muito mais elevada do que aquela a partir de mESCs 26,27. Aqui, um sistema quimicamente definido para a indução neural que foi bem estabelecida por grupo de Zhang 7 foi descrita. Sem a adição de factores exógenos caudalizing, este protocolo eficiente gera precursores telencefálicos de PSCs humanos 27. Esses progenitores pode, então, ser diferenciadas em células progenitoras dorsal ou ventral, regulando a sinalização de Wnt e sonic hedgehog (SHH). Dorsal Os progenitores podem ainda se diferenciar em neurônios e glutamatérgicafficiently 27. Além disso, este protocolo também funciona bem para a geração de neurónios glutamatérgicos do iPSCs humano 28, que permite a geração de neurónios específicos do paciente, que podem ser utilizados para explorar o mecanismo de acção, assim como terapias potenciais para uma grande variedade de doenças . Além disso, o nosso sistema também fornece uma plataforma para explorar o desenvolvimento e especificação de diversos tipos neuronais no telencéfalo.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11330-032</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>1140-050</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (14.3 M)</td>
			<td>Sigma</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine)</td>
			<td>Sigma</td>
			<td>116K5103</td>
		</tr>
		<tr>
			<td>Laminin (human)</td>
			<td>Sigma</td>
			<td>L-6274</td>
		</tr>
		<tr>
			<td>Laminin (mouse)</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini</td>
			<td>100-106</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
		</tr>
		<tr>
			<td>ROCK Inhibitor</td>
			<td>Stemgent</td>
			<td>04-0012</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Stemgent</td>
			<td>04-0024</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075</td>
		</tr>
		<tr>
			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
		</tr>
		<tr>
			<td>Foxg1 antibody</td>
			<td>Dr. Y. Sasai</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoxb4 antibody (1:50)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>I12</td>
		</tr>
		<tr>
			<td>Pax6 antibody (1:5000)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>PAX6</td>
		</tr>
		<tr>
			<td>Nkx2.1 antibody (1:200)</td>
			<td>Chemicon</td>
			<td>MAB5460</td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
		</tr>
		<tr>
			<td>vGLUT1 antibody (1:100)</td>
			<td>Synaptic Systems</td>
			<td>135302</td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-02</td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (GDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-10</td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>PrepoTech Inc.</td>
			<td>100-11</td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma</td>
			<td>D-0260</td>
		</tr>
		<tr>
			<td>Sonic hedgehog (SHH)</td>
			<td>R&amp;D</td>
			<td>1845-SH</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Becton Dickinson (BD)</td>
			<td>352098</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352097</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>BD</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>BD</td>
			<td>353047</td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>BD</td>
			<td>353009</td>
		</tr>
		<tr>
			<td>T75 flasks (untreated)</td>
			<td>BD</td>
			<td>353133</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>BD</td>
			<td>353004</td>
		</tr>
		<tr>
			<td>9'' glass pipetes</td>
			<td>Fisher</td>
			<td>13-678-20D</td>
		</tr>
		<tr>
			<td>Steriflip filters (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
		</tr>
		<tr>
			<td>Stericup filters 1,000 ml (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGPU10RE</td>
		</tr>
		<tr>
			<td>Phase contrast microscope (Observer A1)</td>
			<td>Zeiss</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator (Hera Cell 150)</td>
			<td>Thermo Electron Corporation</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biosafety hood (Sterilgard III Advance)</td>
			<td>The Baker Company</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge (5702 R)</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

the specification of telencephalic glutamatergic neurons from human pluripotent stem cells

Aqui, um procedimento passo a passo para gerar eficientemente telencefálicos neurónios glutamatérgicos de células pluripotentes estaminais humanas (PSCs) tem sido descrita. O processo de diferenciação é iniciada quebrando as PSCs humanos em pedaços que completam-se para formar agregados quando as células são colocadas numa cultura em suspensão. Os agregados são então cultivadas em meio de hESC dias 1-4 para permitir a diferenciação espontânea. Durante este tempo, as células têm a capacidade de se transformar em qualquer uma das três camadas germinais. Dos dias 5-8, as células são colocadas num meio de indução neural para empurrá-los para a linhagem neural. Por volta do dia 8, as células são deixadas a ligar em placas de 6 poços e diferenciar tempo durante o qual a forma de células neuroepiteliais. Estas células neuroepiteliais pode ser isolado no dia 17. As células podem então ser mantidos como neuroesferas até que estejam prontas para serem semeadas em lamelas. Utilizando um meio de base sem quaisquer factores caudalizing, as células neuroepiteliais são specified em precursores telencefálicos, que podem então ser adicionalmente diferenciadas em células progenitoras dorsais e neurónios glutamatérgicos telencefálicos eficientemente. No geral, nosso sistema fornece uma ferramenta para gerar humanos neurônios glutamatérgicos aos pesquisadores para estudar o desenvolvimento desses neurônios e as doenças que os afetam.

Aqui, um protocolo para diferenciar PSCs humanos em telencefálicos neurónios glutamatérgicos através de diversos passos críticos: a formação de agregados de PSC, a indução de células neuroepiteliais, a geração de células progenitoras telencefálicos, e a diferenciação terminal de células progenitoras em neurónios estes telencefálicos (Figura 1) possui foram descritos. Este sistema é robusto e eficiente na geração de células progenitoras telencefálicos e neurónios glutamatérgicos...

Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Este procedimento gera os neurónios telencefálicos atravessando checkpoints que são semelhantes aos observados durante o desenvolvimento humano. As células são deixadas a diferenciar-se espontaneamente, são expostas a factores que os empurram no sentido da linhagem neural, são isolados, e são plaqueadas em lamelas de cobertura para permitir a diferenciação terminal e maturação.

A especificação de telencefálicos neurónios glutamatérgicos de células-tronco pluripotentes humanas

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Research

JoVE Journal

Neuroscience

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

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 ...

Neurociência

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

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

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

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 ...

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

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 ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Método para alta velocidade lesão de estiramento de humanos induzida pluripotentes células-tronco derivadas de neurônios em um formato de 96 poços

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Resposta pós-diferenciação de neurônios derivados de células-tronco pluripotentes humanas para triagem de alto conteúdo de Neuritogênese e maturação de sinapse

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversão de pilhas de haste pluripotentes induzidas humanas (iPSCs) em neurônios de motor espinal e craniano funcionais usando vetores de PiggyBac

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Analisando o Transporte Mitocondrial e a Morfologia em neurônios derivados de células-tronco pluripotentes induzidos por humanos em Paraplegia Espástica Hereditária

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

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

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 ...