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

Dette arbejde blev støttet af tilskud fra National Institutes of Health (R00 AG054616 til Y.A.H. og T32 GM136566 til K.C.), Stanford University School of Medicine og et Siebel Fellowship (tildelt SC). Y.A.H. er en GFL Translational Professor fra Center for Translational Neuroscience i Brown Institute for Translational Sciences.

1. Induktion af humant neuron fra humane pluripotente stamceller
<ol><li>Lentivirus forberedelse (~ 5 dage, detaljeret protokol som beskrevet tidligere16)<ol><li>Plade ~ 1 million HEK293T-celler hver T75-kolbe for at få dem ~ 40% sammenløbende, når de udfører transfektion. Transficere dem med plasmider, der udtrykker tetracyklininducerbart Ngn2 og puromycinresistent gen (PuroR; under samme TetO-promotorkontrol), rtTA og de tre hjælperplasmider pRSV-REV, pMDLg/pRRE og VSV-G (12 μg lentivira...

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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=APOE4+causes+widespread+molecular+and+cellular+alterations+associated+with+Alzheimer's+disease+phenotypes+in+human+iPSC-derived+brain+cell+types.">APOE4 causes widespread molecular and cellular alterations associated with Alzheimer's disease phenotypes in human iPSC-derived brain cell types.</a> Neuron. 98 (6), 1294 (2018).</li><li>TCW, 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=Cholesterol+and+matrisome+pathways+dysregulated+in+human+APOE+&#949;4+glia.">Cholesterol and matrisome pathways dysregulated in human APOE &#949;4 glia.</a> bioRxiv. , (2019).</li><li>Ang, C. 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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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Ud over den fysiske og metaboliske støtte til at stabilisere synapsestrukturerne og lette saltsignalledningen ved myelinering, kan oligodendrocytlinjeceller forme neuronalt aktivitetsmønster via hurtige og dynamiske krydssamtaler med neuroner 5,6,7. Mens oligodendroglial-reaktionerne i AD-patologi oprindeligt blev betragtet som blot sekundære i forhold til inflammation og oxidative belastninger, er der nu lovende beviser, der...

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

Oligodendrocyt-afstamningsceller - herunder oligodendrocytprecursorceller (OPC'er), myeliniserende oligodendrocytter og overgangstyper derimellem - udgør en hovedgruppe af humane hjerneceller1, der aktivt deltager i mange kritiske funktioner for korrekt drift og vedligeholdelse af vores centralnervesystem gennem neural udvikling og aldring 2,3,4 . Mens oligodendrocytter er velkendte for at producere myelin for at lette neuronal aktivitetsoverførsel og understøtte aksonal sundhed i hvidt stof, er OPC'er rigelige (~ 5%) i gråt stof, hvor myelinering er knap og udfører aktivitetsafhængige signalfunktioner til at styre læringsadfærd og hukommelsesdannelse 5,6,7,8 . Hvordan oligodendrogliale celler fungerer og dysfunktion i patogenesen af Alzheimers sygdom (AD) og andre aldersassocierede neurodegenerative tilstande er blevet undervurderet9. Utilstrækkeligheden af et passende modelsystem og mangler i den generelle viden til at styre en eksperimentel vej fremad er hovedårsagerne til denne kløft.
I lyset af de seneste gennembrud inden for udlede humane hjerneceller fra pluripotente stamceller, herunder embryonale stamceller (ES) og inducerede pluripotente stamceller (iPS), er sådanne cellulære modeller i forbindelse med moderne genredigeringsværktøjer opstået som robuste værktøjer til at håndtere den indviklede sammenhæng mellem cellulære interaktioner i hjernen og er i stand til at demonstrere humanspecifikke sygdomsmanifestationer10, 11. I betragtning af at individuelle hjernecelletyper kan udvise forskellige og endda modstridende virkninger i lyset af de samme AD-fremmende forhold12,13, tilbyder denne stamcellemetode unikt celletypespecifik information, der tidligere er blevet savnet ved hjælp af etablerede in vivo- eller in vitro-modeller, der kun giver aggregerede aflæsninger fra samlinger af hjernecelletyper. I det sidste årti er der udviklet et stort antal pålidelige protokoller til at generere humane neuroner fra transdifferentiering af ES / iPS-celler eller direkte konvertering fra andre terminalt differentierede celletyper (f.eks. Fibroblaster)14,15. Især kan anvendelsen af centrale neurogene transkriptionsfaktorer (f.eks. Neurogenin 2, Ngn2)16 på humane pluripotente stamceller generere en homogen population af velkarakteriserede neuronale celletyper til rene kulturer uden behov for cokulturering med gliaceller12,17,18. For inducerede humane oligodendrocytter er der et par offentliggjorte protokoller, der kan generere funktionelle celler, der ligner deres primære modstykker, med en bred vifte af effektivitet og efterspørgsel i tid og ressourcer 19,20,21,22,23,24,25,26,27,28 . Til dato er ingen af disse protokoller blevet anvendt til at undersøge, hvordan oligodendrogliale celler reagerer på og påvirker AD-patogenese.
Her beskriver vi vores forbedrede protokoller for enkelt- og blandede kulturer af humaninducerede neuroner (iN'er) og OPC'er / oligodendrocytter (iOPC'er / iOL'er). Den her beskrevne iN-protokol er baseret på den meget anvendte Ngn2-tilgang16 og har den ekstra funktion at være gliafri. De resulterende iN'er er homogene og ligner meget det kortikale lag 2/3 excitatoriske neuroner med karakteristisk pyramidemorfologi, genekspressionsmønster og elektrofysiologiske træk17,18 (figur 1). For at overvinde nogle af de grundlæggende barrierer i rettet differentiering af pluripotente stamceller har vi udviklet en enkel og effektiv metode til lavdosis dimethylsulfoxid (DMSO) forbehandling29,30 og rapporteret en forbedret tilbøjelighed hos humane ES/iPS-celler til at transdifferentiere til iOPC'er og iOL'er31, baseret på en bredt tilpasset protokol af Douvaras og Fossati 32 . Vi har yderligere forenklet protokollen og indarbejdet en robust differentieringsfremmende forbindelse, clemastin 7,33,34, for at fremskynde processen med oligodendroglial modning. Som et resultat (figur 2) kan iOPC'erne genereres på 2 uger (~ 95% positive for markøren O4) og iOL'erne på fire uger (udtrykker modne markører MBP og PLP1). Interessant nok fandt vi, at iOPC'er alene udskiller en bemærkelsesværdig mængde amyloid-β (Aβ), i overensstemmelse med de uafhængige transkriptomiske data, der viser den rigelige ekspression af amyloidprecursorproteinet (APP) og behandlingsprotease β-sekretase (BACE1) i oligodendrocyt-afstamningsceller35,36. Desuden fremmer vores iN-iOPC-samkultursystem ensheathing af axoner ved MBP-positive iOL-processer og giver betydelig støtte til synapsedannelse (figur 3). Således har de protokoller, vi har beskrevet nedenfor, tekniske og biologiske fordele i forhold til tidligere katalogiserede neuron-oligodendroglia co-dyrkningsmetoder og har et løfte om bedre modellering af neurodegenerationen i AD.

<table>
	<tbody>
		<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

I Alzheimers sygdom (AD) og andre neurodegenerative lidelser er oligodendroglial svigt et almindeligt tidligt patologisk træk, men hvordan det bidrager til sygdomsudvikling og progression, især i hjernens grå stof, forbliver stort set ukendt. Dysfunktionen af oligodendrocyt-afstamningsceller er kendetegnet ved mangler i myelinering og nedsat selvfornyelse af oligodendrocytprecursorceller (OPC'er). Disse to defekter er forårsaget i det mindste delvist af forstyrrelsen af interaktioner mellem neuron og oligodendrocytter langs opbygningen af patologi. OPC'er giver anledning til myeliniserende oligodendrocytter under CNS-udvikling. I den modne hjernebark er OPC'er de vigtigste proliferative celler (bestående af ~ 5% af de samlede hjerneceller) og styrer ny myelindannelse på en neural aktivitetsafhængig måde. Sådan neuron-til-oligodendrocytkommunikation er signifikant undervurderet, især i forbindelse med neurodegenerative tilstande som AD, på grund af manglen på passende værktøjer. I de senere år har vores gruppe og andre gjort betydelige fremskridt for at forbedre de nuværende tilgængelige protokoller til at generere funktionelle neuroner og oligodendrocytter individuelt fra humane pluripotente stamceller. I dette manuskript beskriver vi vores optimerede procedurer, herunder etableringen af et co-kultursystem til at modellere neuron-oligodendrocytforbindelserne. Vores illustrative resultater tyder på et uventet bidrag fra OPC'er / oligodendrocytter til hjernens amyloidose og synapse integritet og fremhæver nytten af denne metode til AD-forskning. Denne reduktionistiske tilgang er et kraftfuldt værktøj til at dissekere de specifikke heterocellulære interaktioner ud af den iboende kompleksitet inde i hjernen. De protokoller, vi beskriver her, forventes at lette fremtidige undersøgelser af oligodendrogliale defekter i patogenesen af neurodegeneration.

Direkte generering af humant inducerede neuroner fra humane pluripotente stamceller Det er meget vigtigt, at de begyndende humane pluripotente stamceller udviser en høj grad af pluripotens for en vellykket generering af iN'er eller iOPC'er / iOL'er. Derfor bør celler farves for specifikke markører, såsom Oct4 og SOX2, inden en af induktionsprotokollerne beskrevet i dette manuskript (figur 1A påbegyndes). Humane H1-celler blev brugt til at opnå inducerede excitatoris...

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

Neuron-glial-interaktionerne i neurodegeneration forstås ikke godt på grund af utilstrækkelige værktøjer og metoder. Her beskriver vi optimerede protokoller til opnåelse af inducerede neuroner, oligodendrocytprecursorceller og oligodendrocytter fra humane pluripotente stamceller og giver eksempler på værdierne af disse metoder til forståelse af celletypespecifikke bidrag i Alzheimers sygdom.

Generering af humane neuroner og oligodendrocytter fra pluripotente stamceller til modellering af neuron-oligodendrocytinteraktioner

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

Research

JoVE Journal

Neuroscience

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Effektiv derivering af humane Neuronal progenitorer og neuroner fra Pluripotente humane embryonale stamceller med småmolekyle Induktion

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The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Specifikationen af ​​Telencephalic glutamaterge neuroner fra humane pluripotente stamceller

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

En cGMP-gældende Expansion Metode til aggregater af humane neurale Stem og stamceller arvet fra pluripotente stamceller eller fostrets hjerne 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 ...

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

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

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