Name: generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions
Uploaded: 2020-11-09T15:00:00
Description: Watch this Scientific Journal Video about Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions at JoVE.com

Questo lavoro è stato sostenuto dalle sovvenzioni del National Institutes of Health (R00 AG054616 a Y.A.H. e T32 GM136566 a K.C.), dalla Stanford University School of Medicine e da una Siebel Fellowship (assegnata a S.C.). Y.A.H. è un professore traslazionale GFL presso il Center for Translational Neuroscience del Brown Institute for Translational Sciences.

1. Induzione di neuroni umani da cellule staminali pluripotenti umane
<ol><li>Preparazione del lentivirus (~5 giorni, protocollo dettagliato come descritto in precedenza16)<ol><li>Piastra ~ 1 milione di cellule HEK293T ogni pallone T75, per averle ~ 40% confluenti durante l'esecuzione della trasfezione. Trasfettarli con plasmidi che esprimono Ngn2 inducibile da tetraciclina e gene resistente alla puromicina (PuroR; sotto lo stesso controllo del promotore TetO), rtTA e i tre plasmidi helper pRSV-...

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H., Park, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+reprogramming+methods+for+generation+of+induced-oligodendrocyte+precursor+cells.">Comparison of reprogramming methods for generation of induced-oligodendrocyte precursor cells.</a> Biomolecules &amp; Therapeutics (Seoul). 25 (4), 362-366 (2017).</li><li>Ehrlich, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+generation+of+oligodendrocytes+from+human+induced+pluripotent+stem+cells+using+transcription+factors.">Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (11), 2243-2252 (2017).</li><li>Rodrigues, G. M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+and+scalable+differentiation+of+human+oligodendrocyte+precursors+from+pluripotent+stem+cells+in+a+3D+culture+system.">Defined and scalable differentiation of human oligodendrocyte precursors from pluripotent stem cells in a 3D culture system.</a> Stem Cell Reports. 8 (6), 1770-1783 (2017).</li><li>Hu, B. Y., Du, Z. W., Li, X. J., Ayala, M., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+oligodendrocytes+from+embryonic+stem+cells:+conserved+SHH+signaling+networks+and+divergent+FGF+effects.">Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects.</a> Development. 136 (9), 1443-1452 (2009).</li><li>Izrael, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+oligodendrocytes+derived+from+embryonic+stem+cells:+Effect+of+noggin+on+phenotypic+differentiation+in+vitro+and+on+myelination+in+vivo.">Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo.</a> Molecular and Cellular Neuroscience. 34 (3), 310-323 (2007).</li><li>Yamashita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+oligodendrocyte+progenitor+cells+from+dissociated+monolayer+and+feeder-free+cultured+pluripotent+stem+cells.">Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.</a> PLoS One. 12 (2), 0171947 (2017).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+oligodendrocyte+progenitor+cells+can+myelinate+and+rescue+a+mouse+model+of+congenital+hypomyelination.">Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination.</a> Cell Stem Cell. 12 (2), 252-264 (2013).</li><li>Chanoumidou, K., Mozafari, S., Baron-Van Evercooren, A., Kuhlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+derived+oligodendrocytes+to+study+myelin+diseases.">Stem cell derived oligodendrocytes to study myelin diseases.</a> Glia. 68 (4), 705-720 (2020).</li><li>Chetty, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+tool+to+improve+pluripotent+stem+cell+differentiation.">A simple tool to improve pluripotent stem cell differentiation.</a> Nature Methods. 10 (6), 553-556 (2013).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transient+DMSO+treatment+increases+the+differentiation+potential+of+human+pluripotent+stem+cells+through+the+Rb+family.">A transient DMSO treatment increases the differentiation potential of human pluripotent stem cells through the Rb family.</a> PLoS One. 13 (12), 0208110 (2018).</li><li>Sambo, D., Li, J., Brickler, T., Chetty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+treatment+of+human+pluripotent+stem+cells+with+DMSO+to+promote+differentiation.">Transient treatment of human pluripotent stem cells with DMSO to promote differentiation.</a> Journal of Visualized Experiments: JoVE. 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Oltre al supporto fisico e metabolico per stabilizzare le strutture sinaptiche e facilitare la conduzione del segnale saltatorio da parte della mielinizzazione, le cellule del lignaggio oligodendrocitario possono modellare il modello di attività neuronale attraverso cross-talk rapidi e dinamici con i neuroni 5,6,7. Mentre nella patologia di AD le risposte oligodendrogliali erano inizialmente considerate semplicemente secondarie...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61778/generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions</a>.&#160;The Representative Results section has been updated.
Figure 3 was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61778/61778fig03.jpg" /> 
Figure 3: Co-culture of iNs and iOPCs.&#160;(A) Representative bright field image of co-cultured iNs and iOPCs at Day 7, showing a proper density for further maturation. (B) Representative immunofluorescence image of iNs and iOPCs co-cultured for 28 days. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Right, a segment of iN axon ensheathed by iOL process (MBP+). (C) Synapse formation assayed in 4-week-old co-cultures. Cells were stained for Synapsin 1 (Syn1, green) and MAP2 (red), and synaptic puncta were quantified by confocal analysis of density along the dendritic segments as described17,18. (D) In our co-cultures of iNs and iOPCs (7 days of co-culturing), the expression of astrocyte markers, ALDHL1 and GFAP, is minimal (top), and the expression of microglia markers, TMEM119, TREM2, and CD33, is not detected (N.D.) by qPCR. The contamination from these two glial cell types is thus excluded. <a href="https://www.jove.com/files/ftp_upload/61778/61778fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61778/61778fig3v2.png" /> 
Figure 3: Co-culture of iNs and iOPCs.&#160;(A) Representative bright field image of co-cultured iNs and iOPCs at Day 7, showing a proper density for further maturation. (B) Representative immunofluorescence image of iNs and iOPCs co-cultured for 28 days. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Right, a segment of iN axon ensheathed by iOL process (MBP+). (C) Synapse formation assayed in 4-week-old co-cultures. Cells were stained for Synapsin 1 (Syn1, green) and MAP2 (red), and synaptic puncta were quantified by confocal analysis of density along the dendritic segments as described17,18. (D) In our co-cultures of iNs and iOPCs (7 days of co-culturing), the expression of astrocyte markers, ALDHL1 and GFAP, is minimal (top), and the expression of microglia markers, TMEM119, TREM2, and CD33, is not detected (N.D.) by qPCR. The contamination from these two glial cell types is thus excluded.&#160;(E) Coculturing iOPC with iN leads to the formation of neuron-OPC synapses. The fluorescence-tagged post-synaptic marker PSD95-mCherry is expressed only in OPCs, and display a diffuse pattern in single cultures (left) but aggregate to form puncta in cocultures (right, indicated by arrows; Tuj1, neuronal marker). (F) The expression of well-characterized oligodendroglial genes that can sense and respond to neuronal activities in the pure cultures of iOPCs at Day 14.&#160;<a href="https://www.jove.com/files/ftp_upload/61778/61778fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The fourth&#160;paragraph was updated from:
Co-culturing of iNs and iOPCs 
This protocol is optimized specifically for co-culturing iNs and iOPCs and allow our real-time monitoring of the inter-cellular communications between these two cell types along the course of neural development. The ideal plating densities for both cell types need to be decided with a series of cell number titration to achieve proper differentiation (Figure 3A). After 4 weeks in co-cultures, the iOPCs are expected to be adequately differentiated into OLs that are positive for specific markers such as MBP and extend processes to ensheath axons (Figure 3B). The co-culture system can robustly boost up the number of synapses, indicating that the iOPCs provide a neuronal support through physical contacts or release of trophic factors (Figure 3C). We can maintain the co-cultures in acceptable health condition for up to 6 weeks and observe that the synapse number and other neuronal attributes plateau around the fifth week. Of note, astrocytes and microglia are not present in our preparations and their absence can be documented by checking the expression of specific markers (Figure 3D).
to:
Co-culturing of iNs and iOPCs 
This protocol is optimized specifically for co-culturing iNs and iOPCs and allow our real-time monitoring of the inter-cellular communications between these two cell types along the course of neural development. The ideal plating densities for both cell types need to be decided with a series of cell number titration to achieve proper differentiation (Figure 3A). After 4 weeks in co-cultures, the iOPCs are expected to be adequately differentiated into OLs that are positive for specific markers such as MBP and extend processes to ensheath axons (Figure 3B). The co-culture system can robustly boost up the number of synapses, indicating that the iOPCs provide a neuronal support through physical contacts or release of trophic factors (Figure 3C). We can maintain the co-cultures in acceptable health condition for up to 6 weeks and observe that the synapse number and other neuronal attributes plateau around the fifth week. Of note, astrocytes and microglia are not present in our preparations and their absence can be documented by checking the expression of specific markers (Figure 3D).&#160;The iOPCs express a good number of well-characterized genes that can potentially respond to and mediate the activity-dependent signals from neighboring neurons, in a paracrine (e.g. neurotrophins and metabolites) and/or a synaptic manner (Figure&#160;3E and 3F).&#160;

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61778/generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions</a>.&#160;The Representative Results section has been updated.

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

Le cellule della linea oligodendrocitaria, comprese le cellule precursori degli oligodendrociti (OPC), gli oligodendrociti mielinizzanti e i tipi di transizione intermedi, costituiscono un gruppo importante di cellule cerebrali umane1 che partecipano attivamente a molte funzioni critiche per il corretto funzionamento e mantenimento del nostro sistema nervoso centrale durante lo sviluppo neurale e l'invecchiamento 2,3,4 . Mentre gli oligodendrociti sono ben noti per la produzione di mielina per facilitare la trasmissione dell'attività neuronale e supportare la salute assonale nella sostanza bianca, le OPC sono abbondanti (~ 5%) nella materia grigia dove la mielinizzazione è scarsa e svolgono funzioni di segnalazione dipendenti dall'attività per governare il comportamento di apprendimento e la formazione della memoria 5,6,7,8 . Il modo in cui le cellule oligodendrogliali funzionano e disagiscono nella patogenesi della malattia di Alzheimer (AD) e di altre condizioni neurodegenerative associate all'età è stato poco studiato9. Le inadeguatezze di un sistema modello appropriato e le carenze nella conoscenza generale per guidare un percorso sperimentale in avanti sono le ragioni principali di questa lacuna.
Alla luce delle ultime scoperte nel derivare cellule cerebrali umane da cellule staminali pluripotenti, tra cui le cellule staminali embrionali (ES) e le cellule staminali pluripotenti indotte (iPS), tali modelli cellulari in combinazione con moderni strumenti di modifica genetica sono emersi come strumenti robusti per gestire l'intricato nesso delle interazioni cellulari nel cervello e sono in grado di dimostrare manifestazioni di malattia specifiche per l'uomo10, 11. Considerando che i singoli tipi di cellule cerebrali possono mostrare effetti distinti e persino contrastanti di fronte alle stesse condizioni che promuovono l'AD12,13, questa metodologia di cellule staminali offre in modo univoco informazioni specifiche sul tipo di cellula che in precedenza sono state perse utilizzando modelli stabiliti in vivo o in vitro che forniscono solo letture aggregate da collezioni di tipi di cellule cerebrali. Nell'ultimo decennio, un buon numero di protocolli affidabili sono stati sviluppati per generare neuroni umani dalla trans-differenziazione di cellule ES/iPS o dalla conversione diretta da altri tipi cellulari terminalmente differenziati (ad esempio, fibroblasti)14,15. In particolare, l'applicazione di fattori chiave di trascrizione neurogenica (ad esempio, neurogenina 2, Ngn2)16 a cellule staminali pluripotenti umane può generare una popolazione omogenea di tipi di cellule neuronali ben caratterizzati per colture pure senza necessità di cocoltura con cellule gliali12,17,18. Per gli oligodendrociti umani indotti, ci sono alcuni protocolli pubblicati che possono generare cellule funzionali molto simili alle loro controparti primarie, con una vasta gamma di efficienza e richiesta di tempo e risorse 19,20,21,22,23,24,25,26,27,28 . Ad oggi, nessuno di questi protocolli è stato applicato per studiare come le cellule oligodendrogliali rispondono e influenzano la patogenesi dell'AD.
Qui, descriviamo i nostri protocolli migliorati per colture singole e miste di neuroni indotti umani (iN) e OPC / oligodendrociti (iOPC / iOL). Il protocollo iN qui descritto si basa sull'approccio Ngn216 ampiamente utilizzato e ha la caratteristica aggiuntiva di essere privo di glia. Gli iN risultanti sono omogenei e molto simili ai neuroni eccitatori dello strato corticale 2/3, con morfologia piramidale caratteristica, pattern di espressione genica e caratteristiche elettrofisiologiche17,18 (Figura 1). Per superare alcune delle barriere fondamentali nella differenziazione diretta delle cellule staminali pluripotenti, abbiamo sviluppato un metodo semplice ed efficace di pre-trattamento a basse dosi di dimetilsolfossido (DMSO)29,30 e riportato una maggiore propensione delle cellule ES / iPS umane a transdifferenziarsi in iOPC e iOLs31, basato su un protocollo ampiamente adattato da Douvaras e Fossati 32 . Abbiamo ulteriormente semplificato il protocollo e incorporato un robusto composto che promuove la differenziazione, clemastina 7,33,34, per accelerare il processo di maturazione oligodendrogliale. Di conseguenza (Figura 2), le iOPC possono essere generate in 2 settimane (~95% positivo per il marcatore O4) e iOL in quattro settimane (esprimendo marcatori maturi MBP e PLP1). È interessante notare che abbiamo scoperto che le iOPC da sole secernono una notevole quantità di amiloide-β (Aβ), coerente con i dati trascrittomici indipendenti che mostrano l'abbondante espressione della proteina precursore dell'amiloide (APP) e della proteasi di trasformazione β-secretasi (BACE1) nelle cellule del lignaggio oligodendrocitario35,36. Inoltre, il nostro sistema di co-coltura iN-iOPC promuove l'inguaina degli assoni mediante processi iOL MBP-positivi e fornisce un supporto significativo per la formazione delle sinapsi (Figura 3). Pertanto, i protocolli che abbiamo dettagliato di seguito hanno vantaggi tecnici e biologici rispetto ai metodi di co-coltura neurone-oligodendroglia precedentemente catalogati e promettono di modellare meglio la neurodegenerazione nell'AD.

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		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>ThermoFisher</td>
			<td>PHG 0266</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>MilliporeSigma</td>
			<td>A9501</td>
			<td></td>
		</tr>
		<tr>
			<td>Clemastine</td>
			<td>MilliporeSigma</td>
			<td>SML0445</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ThermoFisher</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>MilliporeSigma</td>
			<td>D3072</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ScienCell</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>H1 human ES cells</td>
			<td>WiCell</td>
			<td>WA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus</td>
			<td>STEMCELL Technologies</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>ThermoFisher</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A medium</td>
			<td>ThermoFisher</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>ThermoFisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-AA</td>
			<td>R&#38;D Systems</td>
			<td>221-AA-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>VWR</td>
			<td>71002-812</td>
			<td></td>
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			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>TR-1003-G</td>
			<td></td>
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			<td>pRSV-REV</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
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			<td>Puromycin</td>
			<td>ThermoFisher</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Tocris</td>
			<td>4366</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Progenitor Freezing Media</td>
			<td>STEMCELL Technologies</td>
			<td>5838</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff SMADi Neural Induction Kit</td>
			<td>STEMCELL Technologies</td>
			<td>8581</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 triiodothyronine</td>
			<td>MilliporeSigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Tempo-iOlogo: Human iPSC-derived OPCs</td>
			<td>Tempo BioScience</td>
			<td>SKU102</td>
			<td></td>
		</tr>
		<tr>
			<td>TetO-Ng2-Puro</td>
			<td>Addgene</td>
			<td>52047</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
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</table>

generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions

Nella malattia di Alzheimer (AD) e in altri disturbi neurodegenerativi, l'insufficienza oligodendrogliale è una caratteristica patologica precoce comune, ma il modo in cui contribuisce allo sviluppo e alla progressione della malattia, in particolare nella materia grigia del cervello, rimane in gran parte sconosciuto. La disfunzione delle cellule della linea oligodendrocitaria è caratterizzata da carenze nella mielinizzazione e compromissione dell'auto-rinnovamento delle cellule precursori degli oligodendrociti (OPC). Questi due difetti sono causati almeno in parte dall'interruzione delle interazioni tra neurone e oligodendrociti lungo l'accumulo di patologia. Gli OPC danno origine a oligodendrociti mielinizzanti durante lo sviluppo del SNC. Nella corteccia cerebrale matura, le OPC sono le principali cellule proliferative (che comprendono ~ 5% delle cellule cerebrali totali) e controllano la formazione di nuova mielina in modo dipendente dall'attività neurale. Tali comunicazioni neurone-oligodendrocita sono significativamente poco studiate, specialmente nel contesto di condizioni neurodegenerative come l'AD, a causa della mancanza di strumenti appropriati. Negli ultimi anni, il nostro gruppo e altri hanno compiuto progressi significativi per migliorare i protocolli attualmente disponibili per generare neuroni funzionali e oligodendrociti individualmente da cellule staminali pluripotenti umane. In questo manoscritto, descriviamo le nostre procedure ottimizzate, compresa la creazione di un sistema di co-coltura per modellare le connessioni neurone-oligodendrocitario. I nostri risultati illustrativi suggeriscono un contributo inaspettato da OPC / oligodendrociti all'amiloidosi cerebrale e all'integrità delle sinapsi ed evidenziano l'utilità di questa metodologia per la ricerca sull'AD. Questo approccio riduzionista è un potente strumento per sezionare le specifiche interazioni eterocellulari dalla complessità intrinseca all'interno del cervello. I protocolli che descriviamo qui dovrebbero facilitare studi futuri sui difetti oligodendrogliali nella patogenesi della neurodegenerazione.

Generazione diretta di neuroni umani indotti da cellule staminali pluripotenti umane È molto importante che le cellule staminali pluripotenti umane di partenza mostrino un alto grado di pluripotenza per la generazione di successo di iNs o iOPC / iOL. Pertanto, le cellule dovrebbero essere colorate per marcatori specifici, come Oct4 e SOX2, prima di iniziare uno dei protocolli di induzione descritti nel presente manoscritto (Figura 1A). Le cellule H1 umane sono state util...

Watch this Scientific Journal Video about Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions at JoVE.com

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

Le interazioni neurone-glia nella neurodegenerazione non sono ben comprese a causa di strumenti e metodi inadeguati. Qui, descriviamo protocolli ottimizzati per ottenere neuroni indotti, cellule precursori degli oligodendrociti e oligodendrociti da cellule staminali pluripotenti umane e forniamo esempi dei valori di questi metodi nella comprensione dei contributi specifici del tipo di cellula nella malattia di Alzheimer.

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 ...

The protocols described here, are expected to facilitate future studies on oligodendroglial defects, in the pathogenesis of neuro degeneration. This is an improved method that can be used to generate functional human oligodendrocyte lineage cells in only three to four weeks, and establish a co-culture system to model the neuron oligodendrocyte connections. The protocol can be used to study not only Alzheimer's disease, but any other disease associated aspect of neuron oligodendrocyte interactions.To begin, plate approximately one million HEK293T cells in each T75 flask to achieve 40%confluency, then transfect them with plasmids expressing tetracycline inducible NGN2 and puromycin resistant tetracycline transactivator gene, along with the three helper plasmids. Use PEI for transfection. Change the media after 16 hours.Harvest released viral particles by collecting culture media every day and replacing it with fresh media for three days. Pool the collected media for purification, then filter the virus through a 0.22 micrometer filter and centrifuge at 49, 000 times G for 90 minutes. After centrifugation, re-suspend the pellet in the appropriate volume of PBS glucose.Use commercially available H1 human ES cells to perform neuron induction. Culture the cells on extracellular matrix solution coated 6-well plates with ESL maintenance medium and incubate the plates at 37 degrees Celsius with 5%carbon dioxide. Two days before induction of differentiation, detach ES cells by adding one milliliter of cell detachment solution and incubating at room temperature for 10 minutes.Transfer the cells to a tube, then wash the well with two milliliters of media and add it to the same tube. Centrifuge the cells at 300 times G for five minutes. Then re-suspend the pellet in media and plate the cells onto matrix coated 6-well plates at the seating density of one times 10 to the fifth cells per well.On the next day, add lentiviruses, expressing NGN2 plus puro. R, and tetracycline transactivator together with polybrene in fresh ES cell maintenance medium. On day zero, add two micrograms per milliliter doxycycline in DMEM/F-12 medium supplemented with N2 and without morphogens.On day one, add puromycin in fresh DMEM/F-12, plus N2 and doxycycline to the final concentration of one microgram per milliliter. Select the transduced cells in puromycin for at least 24 hours. On day two, detach differentiating neurons with cell detachment solution and re-plate them on 24-well plates coated with matrix solution.Maintain them in MBA B27 medium without doxycycline. Culture pure induced neurons on the plates, coated with extracellular matrix-based solutions. To perform neural progenitor cell generation, culture H1 human ES cells and transdifferentiate them into neural progenitor cells, as described in the text manuscript.On day negative one, seed 0.5 to one million cells in ES cell maintenance medium into each well of a 6-well plate coated with growth factor reduced matrix solution. On day zero, treat cells for 24 hours with ES cell maintenance medium supplemented with 2%DMSO. On days one through six, change the full media with warm neural induction medium containing some add inhibitors from a commercial kit.On day seven, passage NPCs using cell detachment solution and plate them at a seating density of one to two times 10 to the fifth cells per well of a 24-well plate. Assay the differentiation efficiency by immunohistochemical staining for absence of pluripotentcy marker and presence of NPC markers. To generate the oligodendrocyte precursor cells, plate the NPCs in warm neural induction medium with some add inhibitors from a commercial kit.On day eight, prepare a solution of 1%DMSO in the OPC differentiation medium, and treat the plated NPCs for 24 hours. On day nine, replace the media with fresh OPC differentiation medium without DMSO, then feed the cells every other day until day 15. If the the cells reach confluence before day 15, passage them to the seating density of one to two times 10 to the fifth cells per well.On day 14, plate OPCs in OPC differentiation medium at a density of one to two times 10 to the fifth cells per well, in a 24-well plate. Test cells for the presence of OPC specific markers by IHC staining or qPCR, and for the absence of NPC markers. On day 15, replace media with OL maturation medium.Change the medium every other day or every day. When cells reach 90%confluence, split them at a one to three ratio for up to two passages or until cell division slows down substantially. If OPCs divide too fast and reach confluencey in less than three days, add Ara-C at a concentration of two to five micromolar for one to three days.On day 14, plate the IOPCs at a density of one times 10 to the fifth cells per well in a 24-well plate in OPC differentiation medium. On the next day, detach the induced human neurons with cell detachment solution. Add neurons onto the cultured OPCs, plating at the seating density of two times 10 to the fifth cells per well.Use co-culture medium containing neurobasal A medium, 2%B27 supplement, and 100 nanograms per milliliter T3 triiodothyronine. Change the medium on the next day and then every other day. If OPCs proliferate too fast and reach confluency in less than three days, add two to five micromolar Ara-C.It is important that the starting human pluripotent stem cells exhibit a high degree of pluripotentcy for the successful generation of induced neurons, or OPCs and oligodendrocytes. Therefore, cells should be stained for specific markers, such as OCT-4 and SOX2, before starting either of the induction protocols. Human H1 cells were used to obtain induced excitatory forebrain neurons.Pure neurons in culture started expressing synapse in one at day 14 to 16. IOPCs were generated in two weeks and mature oligodendrocytes in four to five weeks. DMSO treatment enriched the number of cells in the early G1 phase for better signaling Integration.Specific OPC markers were detected as early as two weeks after plating of ES H1 cells. The IOPC population at two weeks was fairly homogenous, with over 95%of cells positive for 04 staining and a high level of immunoreactivity for other markers. After initiation of oligodendrocyte maturation, specific oligodendrocyte markers were detected starting at day 28.The ideal plating densities for induced neurons, and IOPCs, need to be determined with a series of cell number titrations to achieve proper differentiation. After four weeks, in co-cultures, the IOPCs should be adequately differentiated into oligodendrocytes that are positive for specific markers such as MBP. This co-culture system can robustly boost the number of synapses, indicating that the IOPCs provide a neuronal support through physical contact or release of trophic factors.This described procedure can be further sophisticated by adding different brain cell types. For example, stem cells derived human astrocytes to model novel aspects of neuron glial interactions in health and disease.

Research

JoVE Journal

Neuroscience

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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

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

Derivazione efficiente di progenitori neuronali umane e Neuroni pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

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

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

La specifica di telencefalici neuroni glutamatergici di cellule staminali umane pluripotenti

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

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

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 ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Derivazione libero-Feeder della cresta neurale progenitrici Cellule pluripotenti da cellule staminali umane

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

Regia neuroni dopaminergici differenziazione da cellule staminali pluripotenti umane

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

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 ...

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

Conversione di cellule staminali pluripotenti indotte dall'uomo (IPSC) in neuroni motore spinale e cranico funzionale utilizzando vettori PiggyBac

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

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

Derivazione, espansione, crioconservazione e caratterizzazione di cellule endoteliali microvascolari cerebrali da cellule staminali pluripotenti indotte dall'uomo

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Creazione di una piattaforma elettrofisiologica per la modellazione della SLA con astrociti e neuroni derivati da cellule staminali pluripotenti umane specifiche a livello regionale

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...