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> Per preparare fiasche rivestite diluire 5 mg di poli-D-lisina (PDL) in 100 ml di acqua deionizzata (DI-acqua), poi cappotto fiasche T75 con 50 ug / ml di PDL a temperatura ambiente (RT) al buio. Dopo 1 ½ ora, aspirare il Pdl e risciacquare i palloni una volta con acqua demineralizzata. Lasciate le fiasche secco prima della semina delle cellule (Tabella 1). </li></ol> 2. Avvio di differenziazione delle cellule progenitrici neurali in oligodendrociti progenitrici derivate (DOP) Il protocollo èOlate cellule progenitrici del SNC è descritto in una precedente pubblicazione e non è parte di questo protocollo 1. Le cellule progenitrici del sistema nervoso centrale sono stati isolati dal telencefalo di un 8 settimane di gestazione il cervello del feto, ottenuto in accordo con le linee guida NIH. <ol><li> Crescere multipotenziale popolazione di cellule progenitrici neurali sul PDL fiasche rivestite in media Neurobasal supplementato con 25 ng / ml di bFGF e 20 ng / ml di fattore di crescita epidermico (EGF) (Figura 1A). Non usarli se maggiore di passaggio 11 (p11). </li><li> Iniziare a differenziare le cellule progenitrici neurali, quando la cultura è di circa il 70% confluenti. </li><li> Rendere terreno completo differenziazione oligodendrociti utilizzando DMEM privo di siero / PROSCIUTTI F12 medie 01:01 supplementato con albumina di siero bovino, L-glutammina, gentamicina, componenti N2, T3, Shh, NT-3, bFGF e PDGF-AA (Tabella 1) . Questo terreno sarà definita come supporto oligo con fattori di crescita (oligo media + GF). </li><li> Rimuovere Progensupporto Itor dai fiaschi, lavare le cellule una volta con tampone fosfato salino (PBS), quindi aggiungere mezzo di oligo + GF (dettagli descritti al punto 2.3) per avviare la differenziazione degli oligodendrociti. Solo le cellule impegnate nella stirpe oligodendrocitario crescerà (Figura 1B). Cultura le cellule in questo mezzo per 3 settimane. </li><li> Ogni settimana, quando le cellule sono a 90-95% di confluenza, passaggio usando tripsina (0,05%)-EDTA (0,1%) (4 ml per una beuta T75). Trasferire il mezzo intero dalle cellule da diversi passaggi in una provetta da 50 ml; questo mezzo condizionato viene utilizzata per estinguere la tripsina. Assicurarsi di non asciugare le cellule. Quindi aggiungere tripsina. </li><li> Incubare le cellule in tripsina per 5 min a RT, delicatamente la beuta volte per aiutare distacco cellulare. </li><li> Quench la tripsina con l&#39;aggiunta di 4-5 mL di terreno condizionato al pallone con le cellule distaccate. </li><li> Trasferire il mezzo e le cellule allo stesso tubo da 50 ml. Centrifugare il mezzo e le cellule a 1.200 giri al minuto (~300 xg) per 5 min a RT. </li><li> Aspirare il surnatante, risospendere il pellet cellulare, con delicatezza ribaltare il tubo, e quindi aggiungere fresche medie oligo + GF. Delicatamente medie pipetta e cellule su e giù per fare una sospensione uniforme cella. </li><li> Contare le cellule, e 2.5e6 trasferimento in ogni nuovo PDL rivestita T75 pallone. </li></ol> 3. Fase finale nella differenziazione degli oligodendrociti <ol><li> Dopo 3 settimane di coltura in oligo media + GF, quando le cellule sono a 70-80% di confluenza, avviare il processo finale di differenziazione (Figura 1C). </li><li> Preparare media nello stesso modo come il mezzo oligo + GF ma senza aggiungere il quattro GF: Shh, NT-3, bFGF e PDGF-AA. Questo mezzo senza il GF quattro saranno definita come supporto di oligo - GF. </li><li> Aspirare il mezzo oligo + GF dal pallone, sciacquare una volta con PBS e quindi aggiungere un volume totale di 16 ml di terreno, di cui ¾ (12 ml) è oligo medio - GF e ¼ (4 ml) è medio PDN condizionato, per 6-10 Giorni). L&#39;uso di mezzo condizionato da PDN non è critico per il processo di differenziazione o la sopravvivenza delle cellule. Abbiamo infatti osservato che solo il contatto diretto con le cellule di PDO PDN in esperimenti di co-coltura ha avuto un effetto sulla durata della sopravvivenza DOP. Il tempo di sopravvivenza medio di oligo - GF poteva essere prolungata per due o tre settimane se le cellule DOP sono co-coltura con cellule PDN. Il protocollo di differenziare cellule progenitrici neurali in PDN è descritto in una precedente pubblicazione e non è parte di questo protocollo 1. La crescita fattore di ritiro dai risultati medi di coltura della cultura progressivamente differenziate di cellule con processi multipli (Figura 1D). </li></ol> 4. Citometria a Flusso del test Il saggio citofluorimetria confronta l&#39;acquisizione di marcatori oligodendrociti durante il processo di differenziazione in rapporto con quelli della popolazione parentale. <ol><li> Usa pro neuralegenitors il controllo. Seme cellule DOP a 2.5x10 6 celle in due palloni T75 rivestite con poli-D-lisina in oligo medie + GF. </li><li> Una settimana dopo, sostituire media con fresca media oligo + GF in fusti come una cultura di controllo per la vitalità delle cellule, mentre altri contenitori avuto oligo medio - GF. </li><li> Dissociare cellule DOP in entrambi i mezzi utilizzare 20 U / ml di papaina e non tripsina. Questo perché papaina ha un effetto più delicato sulle cellule durante la dissociazione preservare la morfologia cellulare. </li><li> Aggiungere 5 ml di soluzione salina bilanciata di Earle (EBSS) in una fiala papaina. Posizionare il flacone a 37 ° C per 10 minuti o fino a quando la papaina è completamente sciolto e la soluzione appare chiara. Questa è la soluzione di papaina utilizzata per dissociare le cellule. </li><li> Aggiungere 0,5 ml di EBSS ad un Deoxyribonuclease I (DNasi) flaconcino (Tabella 1). Mescolare delicatamente. Aggiungere 0,25 ml di questa soluzione nel flacone di papaina disciolto. Questo preparato contiene una concentrazione finale di circa 20 U / ml di papaina e 00,005% DNasi. </li><li> Soluzione luogo papaina (come descritto sopra) sulle cellule e incubare a 37 ° C per 15-30 min, monitorare il distacco delle cellule usando un microscopio. Interrompere la reazione con 5 ml di terreno con o senza GF e centrifugare a 1200 rpm per 7 min. </li><li> Trasferire pellet cellulari da entrambi flaconi in due differenti 5 ml provette di polipropilene e lavarli con normale media fisiologica (NPM: 145 mM NaCl, 5 mM KCl, 1,8 mM CaCl 2, Hepes 10 mM e 10 mM di glucosio) supplementato con 1 mg / ml albumina di siero bovino (BSA NPM +). </li><li> Eseguire superficie immunocolorazione a 4 ° C per 20 minuti usando anticorpi primari specifici e adeguatamente titolata per A2B5, O4, GALC (Tabella 2). </li><li> Lavare le cellule con NPM + BSA a 4 ° C per 5 minuti e incubare con specifici anticorpi secondari a 4 ° C per 20 min (Tabella 2). </li><li> Per macchia antigeni intracellulari tra cui nestina, proteina acida fibrillare gliale (GFAP), classe III e essereta,-tubulina, e la proteina basica della mielina (MBP) (Tabella 2), fissare le cellule in 2% paraformaldeide (PFA) per 20 minuti a RT e permeabilize freddo con etanolo al 70% per 15 min a -20 ° C. </li><li> Lavare le cellule con PBS 1x e incubare con specifici anticorpi secondari (Tabella 2). </li><li> Per discriminare tra cellule intatte e detriti subcellulare durante l&#39;analisi di citometria a flusso, colora le cellule con 4,6-Diamidino-2-phenyllindole, cloridrato (DAPI) (Life Technologies, Grand Island, NY). </li><li> In ottimizzare il protocollo di cui sopra, abbiamo eseguito tutti gli esperimenti di controllo necessarie per confermare la specificità di ogni immunoreagenti. Brevemente, per gli anticorpi marcati direttamente, il controllo appropriato negativo era un anticorpo direttamente coniugato della stessa classe isotipo di immunoglobuline (o sottoclasse) e coniugato con fluorocromo come anticorpo primario (cioè il controllo di isotipo). Per immunostaining indiretto di anticorpi primari, l&#39;appropriato controllo negativo wcome anticorpo secondario coniugato con il fluorocromo di interesse che specificamente mirato alla classe di immunoglobuline (o sottoclasse) del ospite in cui è stato generato l&#39;anticorpo primario. Quando gli anticorpi primari da più host (topo, coniglio, pollo) sono stati utilizzati insieme, abbiamo utilizzato l&#39;anticorpo secondario di mira l&#39;anticorpo primario di ogni host e ogni anticorpo secondario utilizzato è stato in genere cross-adsorbito contro le immunoglobuline dagli altri host per ridurre al minimo cross- ospitare immunoreattività. I controlli positivi inclusi immunostaining diretta o indiretta di preparazioni cellulari note per esprimere gli antigeni di interesse utilizzando sia immunoreazioni diretto o indiretto con gli anticorpi primari specifici per questi antigeni. I metodi di ottimizzazione sopra sono state eseguite una volta per ogni tipo di immunocolorazione esperimenti. Immunoreazioni di controllo ha evidenziato alcuna significativa cross-epitopo immunoreattività tra gli anticorpi primari e secondari. Citometria di flusso è stata eseguita su uniformementesospesi cellule contrassegnati in modo fluorescente utilizzando una cella FACVantage SE sorter (BD Biosciences, San Jose, CA) dotato di tre laser, che forniscono lunghezze d&#39;onda di eccitazione 488 nm sintonizzate, 647 nm, e un ampio UV (351-364 nm) (Figura 2) . </li></ol> 5. Test di immunofluorescenza Nota: Per gli esperimenti di immunofluorescenza, DOP sono placcati in oligo media + GF o oligo medio - GF a 2.5e5 in piastre da 6 pozzetti rivestiti con PDL. Le cellule vengono fissate in 2% PFA in vari momenti dopo il prelievo di fattori di crescita. L&#39;anticorpo macchia viene eseguita su piastre da 6 pozzetti di plastica anziché coprioggetto. <ol><li> Rimuovere il supporto e aggiungere il 2% PFA. Incubare per 10 minuti a RT. Eliminare PFA. Lavare le cellule per 3 volte con PBS, 5 min ciascuna volta. Fare attenzione a non asciugare le cellule. </li><li> Permeabilize cellule per marcatori intracellulari mediante soluzione di Triton 0,25% in PBS per 10 minuti a RT. </li><li> Per prevenire il legame non specifico, cellule blocco fo 10 min con HHG (1 mM tampone HEPES, 2% siero di cavallo, e siero di capra 10% in soluzione salina bilanciata di Hanks &#39;). Diluire specifici anticorpi primari (Tabella 2) per una predeterminata concentrazione HHG. Posizionare anticorpi diluiti su cellule. Incubare per 1 ora a RT l&#39;agitatore o a 4 ° C per una notte. </li><li> Lavare le cellule per 3 volte con PBS per 5 minuti ogni volta. Diluire appropriati anticorpi secondari (Tabella 2) accoppiato con un fluorocromo ad una predeterminata concentrazione HHG. Mettere la miscela di anticorpi secondari diluiti su cellule. Incubare per 1 ora a RT su un agitatore nel buio. </li><li> Lavare le cellule per 3 volte con PBS per 5 minuti ogni volta. </li><li> Per evitare la fotodecolorazione aggiungere 10 ml di prolungare oro con DAPI per le cellule e mettere un coprioggetto di vetro su di essi. Visualizzare le cellule marcate usando un microscopio a fluorescenza Axiovert 200M (Zeiss, Thornwood, NY) (Figura 3). </li></ol>

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Nessun conflitto di interessi dichiarati.

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 ...

Watch this Scientific Journal Video about Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain at JoVE.com

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 ...

progenitor-derived-oligodendrocyte-culture-system-from-human-fetal

Research

JoVE Journal

Neuroscience

14.8K Views.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Primaria, umani fetali di derivazione cerebrale, le cellule progenitrici multipotenti proliferano In vitro 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.

Video: Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Generazione di cellule staminali neurali da scartate tessuto fetale umano corticale

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Ricerca

Neuroscienze

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Derivazione di Arricchito Culture oligodendrociti e degli oligodendrociti / Neuron mielinizzanti co-culture di tessuti murini post-natale

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolamento e coltura di cellule endoteliali dal embrionali Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

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.

Un metodo di espansione cGMP-applicabile per Aggregati di neurali umane staminali e cellule progenitrici derivate da cellule staminali pluripotenti o fetale tessuto cerebrale

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

Immagini di corteccia cerebrale primario celle utilizzando un sistema 2D cultura dal vivo

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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.

Semplice generazione di una cultura ad alto rendimento dei neuroni indotti da fibroblasti umani della pelle 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 ...

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

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used 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 ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissezione e isolamento della glia murina da più regioni del sistema nervoso centrale

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Ottenere Microglia Umana da Tessuto Cerebrale Umano Adulto

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

Generazione di neuroni umani e oligodendrociti da cellule staminali pluripotenti per la modellazione delle interazioni neurone-oligodendrociti

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...