Name: progenitor-derived oligodendrocyte culture system from human fetal brain
Uploaded: 2012-12-20T11:00:00
Description: Watch this Scientific Journal Video about Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain at JoVE.com

Questa ricerca è stata sostenuta dal programma di ricerca intramurale presso il National Institutes of Health, NINDS. Gli autori desiderano ringraziare tutti i membri del Laboratorio di Medicina Molecolare e Neuroscienze, Rick Dreyfuss per aiutare con la microscopia e Pamela Vagliatura C. per l&#39;aiuto con la redazione.

Nota: per la coltura di routine di progenitrici neurali e cellule della linea oligodendrocitario, incubazione avviene a 37 ° C in un umidificata al 5% di CO 2 nell&#39;atmosfera. Ogni 2 giorni, il mezzo viene sostituito con 50 a 100% di mezzo fresco se la cultura è 40-70% confluenti. Al momento della confluenza vicino, le colture sono diversi passaggi a 2-2.5e6/T75 pallone di solito su una pianificazione settimanale. 1. Preparazione del Flask Coated <ol><li> P...

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Questo protocollo descrive come oligodendrociti fetali derivare da cellule primarie umane progenitrici neurali e caratterizzare il loro fenotipo utilizzando sia citometria di flusso e colorazione di immunofluorescenza. L&#39;espansione e la crescita dei progenitori neurali fetali da CNS è stata molto ben descritta 1-4. Tuttavia, ottenere sufficienti oligodendrociti umani per la sperimentazione in vitro resta difficile, anche se è possibile identificare e isolare precursori gliali da materiale umano...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Concentrazione finale </td></tr><tr><td> DME / PROSCIUTTI F12 01:01 </td><td> Omega Scientist </td><td> DM-251 </td><td> 1X </td></tr><tr><td> Albumina bovina </td><td> Sigma </td><td> A9418 </td><td> 1% </td></tr><tr><td> Gentamicina </td><td> Qualità Biologicals </td><td> 120-098-031 </td><td> 50 ug / ml </td></tr><tr><td> L-Glutammina </td><td> Qualità Biologicals </td><td> 118-084-061 </td><td> 2 mM </td></tr><tr><td> T3 </td><td> Sigma </td><td> T2877 </td><td> 3 Nm </td></tr><tr><td> N2 Componenti </td><td> Gibco BRL </td><td> 17502 </td><td> 1:100 </td></tr><tr><td> NT-3 </td><td> Peprotech Inc </td><td> 450-03 </td><td> 2 ng / ml </td></tr><tr><td> Shh </td><td> R &amp; D System </td><td> 1314-SH/CF </td><td> 2 ng / ml </td></tr><tr><td> bFGF </td><td> Peprotech Inc </td><td> 100-18B </td><td> 20 ng / ml </td></tr><tr><td> PDGF-AA </td><td> Peprotech Inc </td><td> 100-13A </td><td> 10 ng / ml </td></tr><tr><td> PDL </td><td> Sigma </td><td> P6407 </td><td> 50 ug / m </td></tr><tr><td> PFA </td><td> Scienze della microscopia elettronica </td><td> 15712 </td><td> 2% </td></tr><tr><td> Tripsina </td><td> Qualità Biologicals </td><td> 118-087-721 </td><td></td></tr><tr><td> Papaina </td><td> Worthington </td><td> LK003178 </td><td> 20 U / ml </td></tr><tr><td> DNasi fiale </td><td> Worthington </td><td> LK003172 </td><td> 0,005% </td></tr><tr><td> EBSS </td><td> Worthington </td><td> LK003188 </td><td></td></tr><tr><td> Prolong Oro conDAPI </td><td> Invitrogen </td><td> P36931 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella 1. Reagenti. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Primaria Abs (tutti a 1 pg / ml) </td><td></td><td> Specie Isotype </td><td> Fonte </td><td> Secondaria Abs </td></tr><tr><td colspan="5"> Citometria a Flusso </td></tr><tr><td> A2B5-Biotina </td><td> Mouse </td><td> IgM </td><td> Regalo J. Nielson </td><td> Streptavidina PETR, Invitrogen, CA </td></tr><tr><td> O4FITC </td><td> Mouse </td><td> IgM </td><td> Regalo da J. Nielson </td><td></td></tr><tr><td> A2B5 </td><td> Mouse </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-FITC, Invitrogen, CA </td></tr><tr><td> O4 </td><td> Mouse </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-FITC, Invitrogen, CA </td></tr><tr><td> GALC </td><td> Mouse </td><td> IgG 3 </td><td> Millipore, MA </td><td> gαm IgG 3-PE, Southern Biotech, AL </td></tr><tr><td> MBP </td><td> Pollo </td><td> IgY </td><td> Millipore, MA </td><td> dαck IgY-AMCA, Jackson Immu., PA </td></tr><tr><td> Nestin </td><td> Mouse </td><td> IgG 1 </td><td> Messam et al. 2000 28 </td><td> gαm IgG 1-PECy5, Invitrogen, CA </td></tr><tr><td> GFAP </td><td> Coniglio </td><td> IgG </td><td> Millipore, MA </td><td> gαrb IgG-PE, Jackson Immu, PA </td></tr><tr><td> βIII tubulina </td><td> Mouse </td><td> IgG 2 </td><td> Covance, CA </td><td> gαm IgG 2 a-PETR, Invitrogen, CA </td></tr><tr><td colspan="6"> Immunocitochimica </td></tr><tr><td> βIII tubulina (1:1,500) </td><td> Mouse </td><td> IgG 2a </td><td> Covance, CA </td><td> gαm IgG 2a-FITC, Invitrogen, CA (1:1,000) </td></tr><tr><td> GFAP (1:1,000) </td><td> Coniglio </td><td> IgG </td><td> Millipore, MA </td><td> gαrb IgG-FITC, Jackson Immu, PA (1:500) </td></tr><tr><td> MBP (1:50) </td><td> Pollo </td><td> IgY </td><td> Millipore, MA </td><td> dαck IgY-FITC, Jackson Immu, PA (1:100) </td></tr><tr><td> O4 (1:100) </td><td> Mouse </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-AF546, Invitrogen, CA (1: 100) </td></tr><tr><td> GALC (1:10) </td><td> Coniglio </td><td> IgG </td><td> Millipore, MA </td><td> gαm IgM-AF750, Invitrogen, CA (1:100) </td></tr></tbody></table> Anticorpi Tabella 2 (Abs) utilizzati per la citometria a flusso e saggi di immunocitochimica coniugati per l&#39;anticorpo: PE, ficoeritrina, PETR, ficoeritrina Texas Red, AMCA, ammino-metil-cumarina-acetato, Cy, cianinico, FITC, isotiocianato di fluorescina Ig:.. Immunoglobuline. . AF: Alexa Fluor; gαm: capra anti-topo; gαrb: capra anti-coniglio; dαck: asino anti-pollo. </td></tr></tbody></table>

progenitor-derived oligodendrocyte culture system from human fetal brain

La differenziazione dei progenitori neurali umane in tipi di cellule neuronali e gliali offre un modello da studiare e confrontare regolazione molecolare dello sviluppo neurale linea cellulare. Espansione in vitro di progenitori neurali da tessuto fetale CNS è stato ben caratterizzato. Nonostante l&#39;identificazione e l&#39;isolamento di progenitori gliali da umano adulto sub-corticale della sostanza bianca e lo sviluppo delle diverse condizioni di coltura per la differenziazione diretta di progenitori neurali fetali in oligodendrociti che producono mielina, l&#39;acquisizione di sufficienti oligodendrociti umani per la sperimentazione in vitro rimane difficile. Differenziazione dei galactocerebroside + (GALC) e O4 + precursori degli oligodendrociti o cellule progenitrici (OPC) da cellule precursori neurali è stato segnalato con secondo cervello fetale trimestre. Tuttavia, queste cellule non proliferano in assenza di cellule di supporto tra astrociti e neuroni, e si perdono rapidamente nel tempo in cultura. Rimane la necessità di un sistema di coltura per produrre cellule della linea oligodendrocyte adatto per la sperimentazione in vitro. Cultura di oligodendrociti primari umani potrebbe, per esempio, essere un modello utile per studiare la patogenesi di neurotropici agenti infettivi come il poliomavirus umano, JCV, che in vivo infetta le cellule. Queste cellule coltivate potrebbe anche fornire modelli di altre malattie demielinizzanti del sistema nervoso centrale (CNS). Primaria, fetale umano derivato dal cervello, le cellule progenitrici multipotenti neurali proliferano in vitro, pur mantenendo la capacità di differenziarsi in neuroni (cellule progenitrici derivate da neuroni, PDN) e astrociti (cellule progenitrici derivate da astrociti, PDA) Questo studio mostra che progenitori neurali può essere indotta a differenziare attraverso molte delle fasi di sviluppo lignaggio oligodendrocitario (progenitrici derivate oligodendrociti, DOP). Noi cellule progenitrici neurali in coltura DMEM-F12 senza siero dei media suptegrato con fattore di crescita dei fibroblasti (bFGF), il fattore di crescita derivato piastrine (PDGF-AA), Sonic hedgehog (Shh), fattore neurotrofico 3 (NT-3), N-2 e triiodotironina (T3). Le cellule coltivate sono diversi passaggi in cellule 2.5e6 per fiaschi 75 centimetri circa ogni sette giorni. Utilizzando tali condizioni, la maggior parte delle cellule in coltura mantenere una morfologia caratterizzata da alcuni processi e marcatori espresse del pre-cellule oligodendrociti, come A2B5 e O-4. Quando rimuoviamo i quattro fattori di crescita (GF) (bFGF, PDGF-AA, Shh, NT-3) e aggiungere mezzo condizionato da PDN, le cellule iniziano ad acquisire più processi e indicatori esprimono specifici di differenziazione degli oligodendrociti, come GALC e la mielina proteina basica (MBP). Abbiamo eseguito la caratterizzazione fenotipica in citometria a flusso multicolore di identificare indicatori unici di oligodendrociti.

E &#39;molto importante per avviare il processo di differenziazione da un 70% -80% coltura confluente di cellule progenitrici neurali (Figura 1A). Molte cellule si estinguerà dopo aver cambiato il mezzo di coltura da capostipite a medio oligo poiché include fattori di crescita specifici. Questo indica che la crescita di cellule progenitrici neurali non impegnati ad un fenotipo oligodendrocitario non sarà supportato dal nuovo mezzo (Figura 1B). Incubazione in mezzo oligo + GF per una ...

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Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Primaria, umani fetali di derivazione cerebrale, le cellule progenitrici multipotenti proliferano<em&gt; In vitro</em&gt; Pur mantenendo la capacità di differenziarsi in neuroni e astrociti. Questo lavoro dimostra che progenitori neurali possono essere indotte a differenziarsi attraverso le fasi della linea oligodendrocitario dal condizionamento con fattori di crescita selezionati.

Progenitore di derivazione Sistema Cultura Oligodendrocyte dal cervello umano fetale

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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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 ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Isolamento dei capillari cerebrali da tessuto cerebrale umano fresco

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Migrazione, chemio-attrazione, e analisi co-cultura per le cellule endoteliali derivate da cellule staminali umane e neuroni GABAergic

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Misurazione e manipolazione dei percorsi neurali funzionalmente specifici nel sistema motorio umano con stimolazione magnetica transcranica

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

Generazione di Oligodendrociti e Mezzo Condizionato oligodendrocito per Esperimenti Co-Culture

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...