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

Ce travail a été soutenu par les subventions des National Institutes of Health (R00 AG054616 à Y.A.H. et T32 GM136566 à K.C.), de la Stanford University School of Medicine et d’une bourse Siebel (attribuée à S.C.). Y.A.H. est professeur translationnel GFL du Center for Translational Neuroscience du Brown Institute for Translational Sciences.

1. Induction de neurones humains à partir de cellules souches pluripotentes humaines
<ol><li>Préparation du lentivirus (~5 jours, protocole détaillé tel que décrit précédemment16)<ol><li>Plaque ~1 million de cellules HEK293T chaque flacon T75, pour les avoir ~40% confluent lors de la transfection. Transfectez-les avec des plasmides exprimant le gène Ngn2 inductible par la tétracycline et résistant à la puromycine (PuroR; sous le même contrôle promoteur TetO), rtTA et les trois plasm...

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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. 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En plus du soutien physique et métabolique pour stabiliser les structures synaptiques et faciliter la conduction du signal salatoire par myélinisation, les cellules de la lignée oligodendrocytaire peuvent façonner le schéma d’activité neuronale via des diaphonies rapides et dynamiques avec les neurones 5,6,7. Alors que dans la pathologie de la MA, les réponses oligodendrogliales étaient initialement considérées comme...

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

Les cellules de la lignée oligodendrocytes, y compris les cellules précurseurs d’oligodendrocytes (OPC), les oligodendrocytes myélinisants et les types transitoires intermédiaires, constituent un groupe important de cellules cérébrales humaines1 qui participent activement à de nombreuses fonctions essentielles au bon fonctionnement et au maintien de notre système nerveux central tout au long du développement neuronal et du vieillissement 2,3,4 . Alors que les oligodendrocytes sont bien connus pour produire de la myéline pour faciliter la transmission de l’activité neuronale et soutenir la santé axonale dans la substance blanche, les OPC sont abondants (~5%) dans la matière grise où la myélinisation est rare et remplissent des fonctions de signalisation dépendantes de l’activité pour régir le comportement d’apprentissage et la formation de la mémoire 5,6,7,8 . Le fonctionnement et le dysfonctionnement des cellules oligodendrogliales dans la pathogenèse de la maladie d’Alzheimer (MA) et d’autres affections neurodégénératives associées à l’âge ont été sous-étudiés9. Les insuffisances d’un système modèle approprié et les lacunes dans les connaissances générales pour guider une voie expérimentale sont les principales raisons de cet écart.
À la lumière des dernières percées dans la dérivation de cellules cérébrales humaines à partir de cellules souches pluripotentes, y compris les cellules souches embryonnaires (SE) et les cellules souches pluripotentes induites (iPS), de tels modèles cellulaires, associés à des outils modernes d’édition de gènes, sont apparus comme des outils robustes pour gérer le lien complexe des interactions cellulaires dans le cerveau et sont capables de démontrer des manifestations de maladies spécifiques à l’homme10, 11. Étant donné que les types individuels de cellules cérébrales peuvent présenter des effets distincts et même contradictoires face aux mêmes conditions favorisant la MA12,13, cette méthodologie de cellules souches offre de manière unique des informations spécifiques au type de cellules qui ont été précédemment manquées à l’aide de modèles établis in vivo ou in vitro qui ne fournissent que des lectures agrégées à partir de collections de types de cellules cérébrales. Au cours de la dernière décennie, un bon nombre de protocoles fiables ont été développés pour générer des neurones humains à partir de la transdifférenciation des cellules ES/iPS ou de la conversion directe à partir d’autres types de cellules différenciées en phase terminale (p. ex. fibroblastes)14,15. En particulier, l’application de facteurs de transcription neurogènes clés (p. ex. neurogénine 2, Ngn2)16 aux cellules souches pluripotentes humaines peut générer une population homogène de types de cellules neuronales bien caractérisées pour des cultures pures sans qu’il soit nécessaire de coculturer avec des cellules gliales12,17,18. Pour les oligodendrocytes humains induits, il existe quelques protocoles publiés qui peuvent générer des cellules fonctionnelles ressemblant fortement à leurs homologues primaires, avec un large éventail d’efficacité et de demande en temps et en ressources 19,20,21,22,23,24,25,26,27,28 . À ce jour, aucun de ces protocoles n’a été appliqué pour étudier comment les cellules oligodendrogliales répondent à la pathogenèse de la MA et l’affectent.
Ici, nous décrivons nos protocoles améliorés pour les cultures uniques et mixtes de neurones induits humains (iNs) et d’OPC / oligodendrocytes (iOPC / iOLs). Le protocole iN décrit ici est basé sur l’approche Ngn2largement utilisée 16, et a la particularité supplémentaire d’être exempt de glie. Les iNs résultants sont homogènes et ressemblent fortement aux neurones excitateurs de la couche corticale 2/3, avec une morphologie pyramidale caractéristique, un modèle d’expression génique et des caractéristiques électrophysiologiques17,18 (Figure 1). Pour surmonter certains des obstacles fondamentaux à la différenciation dirigée des cellules souches pluripotentes, nous avons développé une méthode simple et efficace de prétraitement à faible dose de diméthylsulfoxyde (DMSO)29,30, et rapporté une propension accrue des cellules ES/iPS humaines à se différencier en iOPC et iOLs31, sur la base d’un protocole largement adapté par Douvaras et Fossati 32 . Nous avons encore simplifié le protocole et incorporé un composé robuste favorisant la différenciation, la clémastine 7,33,34, pour accélérer le processus de maturation oligodendrogiale. En conséquence (Figure 2), les OPCi peuvent être générés en 2 semaines (~95% positifs pour le marqueur O4) et les iOLs en quatre semaines (exprimant les marqueurs matures MBP et PLP1). Fait intéressant, nous avons constaté que les CPi seuls sécrètent une quantité remarquable de β amyloïde (Aβ), ce qui correspond aux données transcriptomiques indépendantes montrant l’expression abondante de la protéine précurseur de l’amyloïde (APP) et de la protéase de traitement β-sécrétase (BACE1) dans les cellules de la lignée oligodendrocytaire35,36. De plus, notre système de co-culture iN-iOPC favorise l’engainage des axones par des processus iOL MBP-positifs et fournit un soutien significatif pour la formation de synapses (Figure 3). Ainsi, les protocoles que nous avons détaillés ci-dessous présentent des avantages techniques et biologiques par rapport aux méthodes de co-culture neurone-oligodendroglie précédemment cataloguées, et sont prometteurs pour une meilleure modélisation de la neurodégénérescence dans la MA.

<table>
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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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>pRSV-REV</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<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>
	</tbody>
</table>

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

Dans la maladie d’Alzheimer (MA) et d’autres troubles neurodégénératifs, l’insuffisance oligodendrogliale est une caractéristique pathologique précoce commune, mais la façon dont elle contribue au développement et à la progression de la maladie, en particulier dans la matière grise du cerveau, reste largement inconnue. Le dysfonctionnement des cellules de la lignée oligodendrocytaire est caractérisé par des déficiences dans la myélinisation et une altération de l’auto-renouvellement des cellules précurseurs des oligodendrocytes (OPC). Ces deux défauts sont causés au moins en partie par la perturbation des interactions entre les neurones et les oligodendrocytes tout au long de l’accumulation de la pathologie. Les OPC donnent naissance à des oligodendrocytes myélinisants au cours du développement du SNC. Dans le cortex cérébral mature, les OPC sont les principales cellules prolifératives (comprenant ~ 5% du total des cellules cérébrales) et contrôlent la formation de nouvelles myélines d’une manière dépendante de l’activité neuronale. De telles communications neurone-oligodendrocyte sont significativement sous-étudiées, en particulier dans le contexte de maladies neurodégénératives telles que la MA, en raison du manque d’outils appropriés. Au cours des dernières années, notre groupe et d’autres ont fait des progrès significatifs pour améliorer les protocoles actuellement disponibles pour générer des neurones fonctionnels et des oligodendrocytes individuellement à partir de cellules souches pluripotentes humaines. Dans ce manuscrit, nous décrivons nos procédures optimisées, y compris la mise en place d’un système de co-culture pour modéliser les connexions neurone-oligodendrocytes. Nos résultats illustratifs suggèrent une contribution inattendue des OPC / oligodendrocytes à l’amylose cérébrale et à l’intégrité des synapses et soulignent l’utilité de cette méthodologie pour la recherche sur la MA. Cette approche réductionniste est un outil puissant pour disséquer les interactions hétérocellulaires spécifiques à partir de la complexité inhérente à l’intérieur du cerveau. Les protocoles que nous décrivons ici devraient faciliter les études futures sur les défauts oligodendrogliales dans la pathogenèse de la neurodégénérescence.

Génération directe de neurones induits par l’homme à partir de cellules souches pluripotentes humaines Il est très important que les cellules souches pluripotentes humaines de départ présentent un degré élevé de pluripotence pour une génération réussie d’iNs ou d’iOPCs/iOLs. Par conséquent, les cellules doivent être colorées pour des marqueurs spécifiques, tels que Oct4 et SOX2, avant de commencer l’un ou l’autre des protocoles d’induction décrits dans le présent manuscri...

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

Les interactions neurone-gliale dans la neurodégénérescence ne sont pas bien comprises en raison d’outils et de méthodes inadéquats. Ici, nous décrivons des protocoles optimisés pour obtenir des neurones induits, des cellules précurseurs d’oligodendrocytes et des oligodendrocytes à partir de cellules souches pluripotentes humaines et fournissons des exemples des valeurs de ces méthodes dans la compréhension des contributions spécifiques au type cellulaire dans la maladie d’Alzheimer.

Génération de neurones humains et d’oligodendrocytes à partir de cellules souches pluripotentes pour la modélisation des interactions neurone-oligodendrocytes

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

Génération de cellules souches neurales à partir de tissus humains mis au rebut corticale fœtale

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

Recherche

Neurosciences

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

Dérivation efficace des ressources humaines progéniteurs neuronaux et des neurones à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

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 spécification des neurones glutamatergiques télencéphaliques de cellules souches pluripotentes humaines

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

Une méthode d&#39;extension cGMP applicable pour les agrégats de neurones humains des cellules souches et progénitrices dérivées de cellules souches pluripotentes ou fœtale tissu cérébral

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

Dérivation sans chargeur-de cellules progénitrices de la crête neurale souches pluripotentes humaines cellules

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

Réalisé neurone dopaminergique différenciation de cellules pluripotentes humaines souches

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

Génération simple d’une Culture de haut rendement de neurones induits des fibroblastes de la peau humaine adulte

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

Conversion de cellules souches pluripotentes induites par l’homme (iPSCs) en neurones moteurs spinaux et crânien fonctionnels utilisant des vecteurs 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

Dérivation, expansion, cryopréservation et caractérisation des cellules endothéliales microvasculaires cérébrales à partir de cellules souches pluripotentes induites par l’homme

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

Mise en place d’une plateforme électrophysiologique pour la modélisation de la SLA avec des astrocytes et des neurones dérivés de cellules souches pluripotentes humaines spécifiques à une région

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