This work was supported by the grants from National Institutes of Health (R00 AG054616 to Y.A.H. and T32 GM136566 to K.C.), Stanford University School of Medicine and a Siebel Fellowship (awarded to S.C.). Y.A.H. is a GFL Translational Professor from the Center for Translational Neuroscience in the Brown Institute for Translational Sciences.

1. Human neuron induction from human pluripotent stem cells
<ol>
	<li>Lentivirus preparation (~5 days, detailed protocol as described previously16)
	<ol>
		<li>Plate ~1 million HEK293T cells each T75 flask, to have them ~40% confluent when performing transfection. Transfect them with plasmids expressing tetracycline-inducible Ngn2 and puromycin-resistant gene (PuroR; under the same TetO promoter control), rtTA and the three helper plasmids pRSV-REV, pMDLg/pRRE, and VSV-G (12 &#181;g of lentiviral vector DNA and 6 &#181;g of each of the helper plasmid DNA). Prepare at least three flasks per lentivirus preparation. Use PEI for transfection following the manufacturer&#8217;s instruction. Change the media after 16 h and discard.</li>
		<li>Harvest released viral particles by collecting culture media every day and replace with fresh media for 3 days. Pool the collected media containing viral particles for purification. Filter the virus through a 0.22 &#181;m filter and centrifuge at 49,000 x g for 90 min. Resuspend the pellet in the appropriate volume of PBS-glucose (~150 &#181;L).</li>
	</ol>
	</li>
	<li>Neuron Induction (~5 days) 
	NOTE: This induction protocol (Figure 1A; flow diagram) is highly effective for both iPS and ES cells of validated pluripotency (which can be assayed by immunohistochemistry staining of well-characterized pluripotency markers; Figure 1B).
	<ol>
		<li>Use commercially available H1 human ES cells at the passage of 52 (see Table of Materials). Culture the cells on extracellular matrix solution coated 6-well plates (~0.5 mg of matrix solution per 6-well plate; see Table of Materials) using ES cell maintenance medium (see Table of Materials) and incubate the plates at 37 &#176;C with 5% CO2.</li>
		<li>On Day -2, detach ES cells (80% confluent) with 1 mL of cell detachment solution (see Table of Materials) and incubate at room temperature for 10 min. Transfer the cells to a tube; wash the well with 2 mL of media and combine in the same tube. Centrifuge at 300 x g for 5 min, resuspend the pellet in media, and plate the cells onto matrix coated 6-well plates at the seeding density of 1 x 105 cells per well.</li>
		<li>On Day -1, add lentiviruses expressing Ngn2 plus PuroR and rtTA together with polybrene (8 &#181;g/ml) to the ES cells in fresh ES cell maintenance medium (see Table of Materials). The exact amount of viruses should be determined by actual titers or the titration. We typically add 5 &#181;L each virus per well in a 6-well plate.</li>
		<li>On Day 0, add Doxycycline (2 &#181;g/mL, to activate Ngn2 expression) in DMEM-F12 medium with N2 supplement without morphogens.</li>
		<li>On Day 1, add Puromycin in fresh medium of DMEM-F12 plus N2 and doxycycline, to the final concentration of 1 &#181;g/mL medium. Select the transduced cells in Puromycin for at least 24 h. Higher Puromycin concentration (up to 5 &#181;g/mL) and longer selection period (up to 48 h) may be required to adequately remove the under-transduced cells if the virus titer is low.</li>
		<li>On Day 2, detach differentiating neurons with cell detachment solution (see Table of Materials), and re-plate them on 24-well plates (between 80,000&#8211;200,000 cells/well) coated with matrix solution (see Table of Materials), and maintain them in NBA/B27 medium without doxycycline. The seeding density is critical.</li>
		<li>At this stage, detached neurons can be frozen in specialized commercial freezing medium (see Table of Materials) and stored in liquid nitrogen for up to 3 months. Pure neurons can be plated accounting for the typical ~15%&#8211;20% cell death post-thaw, cultured alone or co-cultured with other brain cell types (see step 3.2.3. for co-culturing with OPCs).</li>
		<li>Culture pure iNs on the plates coated with extracellular matrix-based solutions as instructed by the manufacturer (see Table of Materials). The characteristic pyramidal morphology should be apparent by Day 4 (and Day 6; Figure 1C). The synapse formation can be detected as early as Day 14 to 16 and is prominent at Day 24 by immunohistochemical staining with standard pre- and post-synaptic markers. (Figure 1D; labeled with the pre-synaptic marker Synapsin 1 and the dendritic marker Map2).</li>
	</ol>
	</li>
</ol>
2. Human oligodendrocyte precursor cell (OPCs) induction from pluripotent stem cells and oligodendrocyte maturation
<ol>
	<li>Neural Progenitor Cell (NPC) generation: monolayer protocol (~7 days). See Figure 2A for the flow diagram.
	<ol>
		<li>Culture H1 human ES cells as described earlier (see step 1.2.1.) and trans-differentiate them into neural progenitor cells (NPCs) by an established approach called dual SMADi, with small molecule inhibitors for multiple signaling pathways. Here we use a widely accepted commercial kit and follow the monolayer protocol provided by the manufacturer (see Table of Materials).</li>
		<li>On Day -1, plate 0.5&#8211;1 x 106 cells per well in a 6-well plate coated by a growth factor reduced matrix solution (see Table of Materials; ~0.5 mg of matrix solution per 6-well plate) with ES cell maintenance medium (see Table of Materials). This growth factor reduced matrix solution is used to coat all the plates that will be used in the following steps.</li>
		<li>On Day 0, treat cells for 24 h with ES cell maintenance medium (see Table of Materials) supplemented by 2% DMSO.</li>
		<li>On Day 1&#8211;6, change the full media with warm (37 &#176;C) neural induction medium containing the SMAD inhibitors from the commercial kit (see Table of Materials). If cells divide and reach confluence before Day 7, passage them to the seeding density of 0.5&#8211;1 x 106, as described earlier in step 2.1.2.</li>
		<li>On Day 7, passage NPCs using cell detachment solution (see Table of Materials) and plate at a seeding density of 1&#8211;2 x 105 cells/well of a 24-well plate.</li>
		<li>Assay the differentiation efficiency by immunohistochemical (IHC) staining for absence of pluripotency marker, OCT4 for example, and presence of NPC markers such as PAX6, Nestin, and Sox1.</li>
		<li>At this stage, detached NPCs can be frozen in the specialized commercial NPC freezing media (see Table of Materials) and stored in liquid nitrogen for up to 3 months. After freeze-and-thaw for once, NPCs still retain the multipotency to give rise to neurons, astrocytes, and OPCs with reliable protocols.</li>
	</ol>
	</li>
	<li>Oligodendrocyte precursor cell (OPC) generation (~7 days). Please see Figure 2A for the flow diagram.
	<ol>
		<li>On Day 7, passage NPCs using cell detachment solution (see Table of Materials) and plate them at a seeding density of 1&#8211;2 x 105 cells per well in a 24-well plate in warm (37 &#176;C) neural induction medium plus SMAD inhibitors from the commercial kit (see Table of Materials).</li>
		<li>On Day 8, prepare a solution of 1% DMSO in the OPC differentiation medium and treat the plated NPCs for 24 h. The OPC differentiation medium is composed of: DMEM/F12 medium, 1% N2 supplement, 1% B27 supplement, bFGF at 20 ng/mL, SAG at 1 &#181;M, PDGF-AA at 10 ng/mL (see Table of Materials).</li>
		<li>On Day 9, replace media with fresh OPC differentiation medium without DMSO. Feed the cells every other day until Day 15. If the cells reach confluence before Day 15, passage them to the seeding density of 1&#8211;2 x 105 cells per well as described in step 2.2.1.</li>
		<li>On Day 14, plate OPCs in OPC differentiation medium at a density of 1&#8211;2 x 105 cells/well in a 24-well plate.</li>
		<li>At this stage (Day 15), test cells for the presence of OPC-specific markers by IHC staining or qPCR (e.g., O4, Olig1/2, CSPG4/Ng2, NKX2.2, PDGFRa; Figure 2B) and for the absence of NPC markers (Pax6 or Nestin; Figure 2D). We typically detect the O4 immunoreactivity in more than 95% of the cells at Day 15. Of particular relevance to Alzheimer&#8217;s disease, the expression of APP (amyloid precursor protein), BACE1 (the processing protease &#946;-secreatase 1), and peptide amyloid-&#946; (A&#946;) is abundant in OPCs (Figure 2F).</li>
	</ol>
	</li>
	<li>Oligodendrocyte (OL) maturation (~7&#8211;20 days)
	<ol>
		<li>On Day 15, replace media with OL maturation medium: Neurobasal-A medium, 2% B27 supplement, 1 &#181;M cAMP, 200 ng/mL T3 triiodothyronine, and Clemastine of 1 &#181;M (see Table of Materials). Change the medium every other day or every day, if necessary.</li>
		<li>When cells reach 90% confluence, split at a 1:3 ratio up to 2 passages or until cell division slows down substantially. If OPCs divide too fast and reach confluency in less than 3 days, add Ara-C (see Table of Materials) at a concentration of 2&#8211;5 &#181;M for 1&#8211;3 days. Active proliferation indicates lowered maturation efficiency.</li>
		<li>Examine the efficiency of oligodendroglial maturation by assessing the expression of OL markers, e.g., CLDN11, PLP1, MBP by qPCR, IHC staining or immunoblotting. The characteristic morphology of highly complex structures (Figure 2C) and the expression of OL markers (Figure 2E) should be readily detected by Day 28.</li>
	</ol>
	</li>
</ol>
3. Co-culturing of human induced neurons (iNs) and oligodendrocyte precursor cells (iOPCs)
<ol>
	<li>iOPC plating (~3 days)
	<ol>
		<li>Plate iOPCs at Day 14 at a density of 1 x 105 cells per well in a 24-well plate (as described above in step 2.2.4.) in OPC differentiation medium (as described in step 2.2.2.).</li>
	</ol>
	</li>
	<li>iN-iOPC co-culture set up
	<ol>
		<li>On Day 15, detach the induced human neurons at the step of Day 2 after the Puromycin selection (as described in step 1.2.6.) with cell detachment solution (see Table of Materials).</li>
		<li>Add neurons onto the cultured OPCs, plating at the seeding density of 2 x 105 cells per well in the 24-well plate with growing OPCs (from step 3.1.1). Use the co-culture medium containing Neurobasal-A medium, 2% B27 supplement, and 100 ng/mL T3 triiodothyronine. Change the medium on the next day and then every other day afterwards. If OPCs proliferate too fast and reach confluency in less than 3 days, add Ara-C at a concentration of 2&#8211;5 &#181;M. A representative image of the iNs and iOPCs grown in co-culture after 7 days neurons is shown in Figure 3A.</li>
		<li>Use frozen neurons prepared as described above in step 1.2.7 for co-culturing with OPCs. Plate freeze-and-thaw neurons at a higher density of 3 x 105 cells per well.</li>
		<li>After Day 14&#8211;16 in co-cultures, the synapse formation in iNs can be observed by IHC staining of pre- and post-synaptic markers, and by Day 21 the synaptic puncta should be abundant (Figure 3C) and neuronal activities can be reliably recorded.</li>
		<li>Starting at Day 21, test cells for OL specific markers (for example, MBP and PLP1). By Day 28, we normally observe the phenomenon ensheathing of iN axons by iOL processes, labeled by IHC staining for specific markers (Figure 3B; neurofilament NF for iN axons and MBP for iOPC processes).</li>
	</ol>
	</li>
</ol>

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(149), (2019).</li><li>Douvaras, P., Fossati, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+isolation+of+oligodendrocyte+progenitor+cells+from+human+pluripotent+stem+cells.">Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells.</a> Nature Protocols. 10 (8), 1143-1154 (2015).</li><li>Mei, F., 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=Micropillar+arrays+as+a+high-throughput+screening+platform+for+therapeutics+in+multiple+sclerosis.">Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.</a> Nature Medicine. 20 (8), 954-960 (2014).</li><li>Madhavan, 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=Induction+of+myelinating+oligodendrocytes+in+human+cortical+spheroids.">Induction of myelinating oligodendrocytes in human cortical spheroids.</a> Nature Methods. 15 (9), 700-706 (2018).</li><li>Zhang, Y., 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=Purification+and+characterization+of+progenitor+and+mature+human+astrocytes+reveals+transcriptional+and+functional+differences+with+mouse.">Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse.</a> Neuron. 89 (1), 37-53 (2016).</li><li>Grubman, A., 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+single-cell+atlas+of+entorhinal+cortex+from+individuals+with+Alzheimer's+disease+reveals+cell-type-specific+gene+expression+regulation.">A single-cell atlas of entorhinal cortex from individuals with Alzheimer's disease reveals cell-type-specific gene expression regulation.</a> Nature Neuroscience. 22 (12), 2087-2097 (2019).</li><li>Goldman, S. A., Kuypers, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+an+oligodendrocyte.">How to make an oligodendrocyte.</a> Development. 142 (23), 3983-3995 (2015).</li><li>Behrendt, G., 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=Dynamic+changes+in+myelin+aberrations+and+oligodendrocyte+generation+in+chronic+amyloidosis+in+mice+and+men.">Dynamic changes in myelin aberrations and oligodendrocyte generation in chronic amyloidosis in mice and men.</a> Glia. 61 (2), 273-286 (2013).</li><li>Patzke, 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=Neuromodulator+signaling+bidirectionally+controls+vesicle+numbers+in+human+synapses.">Neuromodulator signaling bidirectionally controls vesicle numbers in human synapses.</a> Cell. 179 (2), 498-513 (2019).</li><li>Piao, 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=Human+embryonic+stem+cell-derived+oligodendrocyte+progenitors+remyelinate+the+brain+and+rescue+behavioral+deficits+following+radiation.">Human embryonic stem cell-derived oligodendrocyte progenitors remyelinate the brain and rescue behavioral deficits following radiation.</a> Cell Stem Cell. 16 (2), 198-210 (2015).</li><li>Keirstead, H. 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+embryonic+stem+cell-derived+oligodendrocyte+progenitor+cell+transplants+remyelinate+and+restore+locomotion+after+spinal+cord+injury.">Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.</a> Journal of Neuroscience. 25 (19), 4694-4705 (2005).</li><li>Kim, D. 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=Rapid+generation+of+OPC-like+cells+from+human+pluripotent+stem+cells+for+treating+spinal+cord+injury.">Rapid generation of OPC-like cells from human pluripotent stem cells for treating spinal cord injury.</a> Experimental &amp; Molecular Medicine. 49 (7), 361 (2017).</li></ol>

The authors have nothing to disclose.

In addition to the physical and metabolic support to stabilize the synapse structures and to facilitate the saltatory signal conduction by myelination, oligodendrocyte lineage cells can shape neuronal activity pattern via rapid and dynamic cross-talks with neurons5,6,7. While in AD pathology the oligodendroglial responses were initially regarded as merely secondary to inflammation and oxidative stresses, there is now promising e...

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

Oligodendrocyte lineage cells&#8212;including oligodendrocyte precursor cells (OPCs), myelinating oligodendrocytes, and transitional types in between&#8212;constitute a major group of human brain cells1 that actively participate in many critical functions for the proper operation and maintenance of our central nervous system throughout neural development and aging2,3,4. While oligodendrocytes are well known for producing myelin to facilitate neuronal activity transmission and support axonal health in white matter, OPCs are abundant (~5%) in gray matter where myelination is scarce and perform activity-dependent signaling functions to govern learning behavior and memory formation5,6,7,8. How oligodendroglial cells function and dysfunction in the pathogenesis of Alzheimer&#8217;s disease (AD) and other age-associated neurodegenerative conditions has been understudied9. The inadequacies of an appropriate model system and deficiencies in general knowledge to guide an experimental path forward are the major reasons for this gap.
In light of the latest breakthroughs in deriving human brain cells from pluripotent stem cells including embryonic stem (ES) and induced pluripotent stem (iPS) cells, such cellular models in conjunction with modern gene editing tools have emerged as robust tools to handle the intricate nexus of cellular interactions in the brain, and are capable of demonstrating human-specific disease manifestations10,11. Considering that individual brain cell types can exhibit distinct and even conflicting effects in the face of the same AD-promoting conditions12,13, this stem cell methodology uniquely offers cell type-specific information that has previously been missed using established in vivo or in vitro models that only provide aggregate readouts from collections of brain cell types. In the last decade, a good number of reliable protocols have been developed to generate human neurons from trans-differentiation of ES/iPS cells or direct conversion from other terminally differentiated cell types (e.g., fibroblasts)14,15. In particular, the application of key neurogenic transcription factors (e.g., neurogenin 2, Ngn2)16 to human pluripotent stem cells can generate a homogeneous population of well-characterized neuronal cell types for pure cultures without a need for coculturing with glial cells12,17,18. For induced human oligodendrocytes, there are a few published protocols that can generate functional cells highly resembling their primary counterparts, with a wide range of efficiency and demand in time and resources19,20,21,22,23,24,25,26,27,28. To date, none of these protocols have been applied to investigate how oligodendroglial cells respond to and affect AD pathogenesis.
Here, we describe our improved protocols for single and mixed cultures of human induced neurons (iNs) and OPCs/oligodendrocytes (iOPCs/iOLs). The iN protocol described here is based on the widely used Ngn2 approach16, and has the additional feature of being glia-free. The resultant iNs are homogenous and highly resemble the cortical layer 2/3 excitatory neurons, with characteristic pyramidal morphology, gene expression pattern and electrophysiological features17,18 (Figure 1). To overcome some of the fundamental barriers in directed differentiation of pluripotent stem cells, we have developed a simple and effective method of low-dose dimethyl sulfoxide (DMSO) pre-treatment29,30, and reported an enhanced propensity of human ES/iPS cells to transdifferentiate into iOPCs and iOLs31, based on a widely-adapted protocol by Douvaras and Fossati32. We have further simplified the protocol and incorporated a robust differentiation-promoting compound, clemastine7,33,34, to accelerate the process of oligodendroglial maturation. As a result (Figure 2), the iOPCs can be generated in 2 weeks (~95% positive for the marker O4) and iOLs in four weeks (expressing mature markers MBP and PLP1). Interestingly, we found iOPCs alone secrete a remarkable amount of amyloid-&#946; (A&#946;), consistent with the independent transcriptomic data showing the abundant expression of the amyloid precursor protein (APP) and the processing protease &#946;-secretase (BACE1) in oligodendrocyte lineage cells35,36. Moreover, our iN-iOPC co-culture system promotes the ensheathing of axons by MBP-positive iOL processes and provides significant support for synapse formation (Figure 3). Thus, the protocols we have detailed below have technical and biological advantages over previously catalogued neuron-oligodendroglia co-culturing methods, and hold a promise in better modeling the neurodegeneration in 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&amp;amp;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

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

Direct generation of human induced neurons from human pluripotent stem cells 
It is very important that the starting human pluripotent stem cells exhibit a high degree of pluripotency for successful generation of iNs or iOPCs/iOLs. Therefore, cells should be stained for specific markers, such as Oct4 and SOX2, before starting either of the induction protocols described in the present manuscript (Figure 1A). Human H1 cells were used to obtain induced excitatory forebrain...

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

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

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

generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells

Research

JoVE Journal

Neuroscience

4.7K Views.  Brown University. De neuron-gliale interacties bij neurodegeneratie worden niet goed begrepen als gevolg van ontoereikende hulpmiddelen en methoden. Hier beschrijven we geoptimaliseerde protocollen om geïnduceerde neuronen, oligodendrocytenvoorlopercellen en oligodendrocyten te verkrijgen uit menselijke pluripotente stamcellen en geven we voorbeelden van de waarden van deze methoden bij het begrijpen van celtypespecifieke bijdragen bij de ziekte van Alzheimer.

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

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Developmental Biology

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

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

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

Medicine

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

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

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

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

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

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Source: Assetta, B.,et al. <a href="https://app.jove.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons&#39; axons.

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days)
	
		Plate ~1 million Human embryonic kidney 293T (HEK293T) ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Differentiating Human Embryonic Stem Cells into Oligodendrocytes

Source: Assetta, B., et al. <a href="https://app.jove.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp (2020).
This video demonstrates the process of generating oligodendrocyte precursor cells (OPCs) from human embryonic stem cells, followed by the maturation of OPCs to oligodendrocytes. This process of generating oligodendrocytes opens numerous avenues to improve research and treatment of neurodegenerative diseases, including Alzheimer&#39;s.

1. Human oligodendrocyte precursor cell (OPC) induction from pluripotent stem cells and oligodendrocyte maturation

	Neural Progenitor Cell (NPC) ...

Advanced Neuronal Models

Generation of Spheroids from Human Pluripotent Stem Cells

Source: Shaker, M. R., et al. <a href="https://app.jove.com/v/63714">Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.</a> J. Vis. Exp. (2022).
This video demonstrates the transformation of human pluripotent stem cells (hPSCs) into spherical brain organoids. By using specific inhibitors, the hPSCs are guided to differentiate towards a neuronal lineage, forming neuroectodermal colonies. Special growth factors are then introduced, which encourage the self-organization and proliferation of the colonies, ultimately leading to the formation of spherical brain organoids.

1. Cortical brain organoid generation (Figure 1) 
NOTE: All the steps in this section of the protocol will occur in a Class 2 biosafety hood unless stated ...

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

Samenvatting

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