Supported by MIUR National Technology Clustersproject IRMI (CTN01_00177_888744), and Regione Emilia-Romagna, Mat2Rep, POR-FESR 2014-2020.
Special thanks to IRET Foundation for hosting the experimental work.

All animal protocols described herein were carried out according to European Community Council Directives (86/609/EEC) and comply with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.
1. Solutions and reagents
<ol>
	<li>Prepare standard medium: DMEM/F12 GlutaMAX 1x; 8 mmol/L HEPES; 100 U/100 &#956;g Penicillin/Streptomycin (1% P/S); 1x B27; 1x N-2.</li>
	<li>Prepare neurosphere medium: add 10 ng/mL bFGF; 10 ng/mL EGF to standard medium.</li>
	<li>Prepare oligosphere/OPC medium: add 10 ng/mL bFGF; 10 ng/mL PDGF-AA to standard medium.</li>
	<li>Prepare oligodendroctye differentiation medium: add 50 nM T3; 10 ng/mL CNTF; 1x N-acetyl-L-cysteine (NAC) to standard medium.</li>
	<li>Prepare non-enzymatic dissociation buffer: add 1% P/S to non-enzymatic dissociation buffer and keep ice cold.</li>
	<li>Prepare sucrose solution: HBSS, 0.3 g/mL sucrose.</li>
	<li>Prepare BSA washing solution: EBSS, 40 mg/mL BSA, 0.02 mL/l HEPES.</li>
	<li>Prepare enzymatic dissociation buffer: HBSS, 5.4 mg/mL D-glucose, 15 mmol/L HEPES, 1.33 mg/mL Trypsin, 0.7 mg/mL Hyaluronidase, 80 U/mL DNase.</li>
	<li>Prepare cytokine mix: TGF-&#946;1, TNF-&#945;, IL-1&#946;, IL-6, IL-17, and IFN-&#947; (20 ng/mL each).</li>
	<li>Prepare cytokine mix vehicle: 0.04% of the stock (10% glycerol/100 nM glycine/25 nM Tris, pH 7.3).</li>
	<li>Prepare oxygen-glucose deprivation medium: standard medium using DMEM w/o glucose.&#160;Depending on the stringency of the desired glucose deprivation condition, it is possible to remove also B27 and/or N2 from the medium to avoid glucose-related compounds (e.g., D-Galactose in B27).</li>
</ol>
2. Dissection and NSC isolation
NOTE: Fetal and adult NSCs were isolated from E13.5 fetal forebrain or 2.5-month-old adult sub-ventricular zone (SVZ), following the Ahlenius and Kokaia protocol9 with modifications.
<ol>
	<li>Fetal NSC cultures 
	NOTE: Before starting the dissections, prepare 1.5 mL tubes containing 150 &#181;L of non-enzymatic dissociation buffer each; clean Petri dishes and add ice cold HBSS.
	<ol>
		<li>Collect the embryos at E13.5 - 14.5 from timed pregnant mice and place in a Petri dish containing cold HBSS.</li>
		<li>Decapitate the embryos using forceps.</li>
		<li>Place the heads of the embryos in a clean Petri dish containing ice cold PBS and remove the skin from the skull with forceps, using magnifying glasses or a stereoscope.</li>
		<li>Once the brain is visible and cleared of skin, squeeze it out by applying pressure at the sides with forceps.</li>
		<li>Remove the cerebellum, keep only the forebrain and remove the meninges with forceps.</li>
		<li>Place the isolated tissue in the non-enzymatic dissociation buffer and repeat the dissection steps with the other embryos. Insert the tissue from 2&#8211;3 animals into each tube containing the buffer.</li>
		<li>Incubate at 37 &#176;C for 15 min under continuous shaking.</li>
		<li>After incubation, add 850 &#181;L of standard medium and mix by pipetting until the suspension is free of clumps.</li>
		<li>If non-dissociated tissue is still visible, wait for 2 min at RT until it deposits at the bottom of the tube.</li>
		<li>When dissociation is complete, count the cells and plate them in suspension at a density of 10&#8211;50 cells/&#181;L in a T-25 or T-45 flask containing 10&#8211;30 mL of neurosphere medium, kept in a vertical position to avoid cell adhesion.</li>
	</ol>
	</li>
	<li>Adult NSC cultures
	<ol>
		<li>Sacrifice animals by cervical dislocation.</li>
		<li>Collect brains from 4&#8211;5 mice in a 50 mL tube containing ice cold HBSS.</li>
		<li>Place the brain on a cold sterile surface. For this purpose, use a T-25 flask filled with water and placed at -20 &#176;C overnight. At the time of the experiment, cover the flask with sterile aluminum foil.</li>
		<li>Place the brain ventral side downwards, in rostro-caudal direction, and remove the olfactory bulbs using a razor blade.</li>
		<li>Using a razor blade, cut 2&#8211;3 coronal slices of 1 mm thickness, from the cortex to the optical chiasma.</li>
		<li>Place the slices on the cold surface in a ventro-dorsal position and identify the corpus callosum and the two lateral ventricles.</li>
		<li>Using magnifying glasses or a stereoscope, isolate the walls of the lateral ventricles, taking care not to carry pieces of the corpus callosum.</li>
		<li>Put the isolated tissue in the enzymatic dissociation buffer (5&#8211;10 mL) and incubate at 37 &#176;C for 15 min.</li>
		<li>Mix the solution, pipetting several times (at least 50), and incubate again at 37 &#176;C for 10 min.</li>
		<li>Neutralize the trypsin by adding 5 mL of standard culture medium and filter the solution using a 70 &#181;m filter.</li>
		<li>Centrifuge the filtered solution for 5 min at 400 x g.</li>
		<li>Resuspend the pellet in the sucrose solution and centrifuge for 10 min at 500 x g.</li>
		<li>Resuspend the pellet in BSA washing solution and centrifuge for 7 min at 400 x g.</li>
		<li>Resuspend the pellet in the standard culture medium, count the cells, and perform plating as described above (in step 2.1.10).</li>
	</ol>
	</li>
</ol>
3. Primary neurospheres
<ol>
	<li>Add the growth factors (bFGF/EGF) every 2 days.</li>
	<li>Every 4&#8211;6 days (depending on cell density), change half of the medium as follows:
	<ol>
		<li>Transfer the entire cell suspension to a 15 or 50 mL tube.</li>
		<li>Centrifuge for 5 min at 400 x g.</li>
		<li>Remove half of the volume.</li>
		<li>Add the same amount of fresh medium, gently mix by pipetting, and add growth factors.</li>
	</ol>
	</li>
</ol>
4. Oligospheres
NOTE: Oligodendrocyte differentiation is performed following the Chen protocol10 with modifications.
<ol>
	<li>When the neurospheres reach a diameter of 100&#8211;150 &#181;m, they are ready to be passed. To do so, transfer the entire cell suspension to a 15 or 50 mL tube, and centrifuge for 5 min at 400 x g.
	<ol>
		<li>Rapidly evaluate the diameter by taking pictures of the spheres using an inverted transmitted light microscope and opening them by ImageJ software.</li>
		<li>Click on the Analyze menu and from the Tools window, select Scale bar.</li>
		<li>Set 150 &#181;m as Width in microns and compare the scale bar with the spheres.</li>
	</ol>
	</li>
	<li>Remove the entire volume by inversion and resuspend the pellet in 180 &#181;L of fresh standard culture medium. Pipette 50 times to allow disaggregation of the spheres.</li>
	<li>Add 810 &#181;L of fresh standard culture medium, count the cells, and re-plate them as described for the neurospheres.</li>
	<li>Add bFGF/PDGF-AA 10 ng/mL every 2 days.</li>
	<li>Every 4&#8211;6 days (depending on cell density), change half of the medium as follows:</li>
	<li>Transfer the entire cell suspension to a 15 or 50 mL tube.</li>
	<li>Centrifuge for 5 min at 400 x g.</li>
	<li>Remove half of the volume.</li>
	<li>Add the same amount of fresh medium, gently mix by pipetting, and add growth factors.</li>
</ol>
5. Plate coating
<ol>
	<li>Poly-D,L-ornithine/laminin coating: at least 2 days before plating the OPCs, add 50 &#181;g/mL poly-D,L-ornithine solution, diluted in PBS, to each well (40 &#181;L/well for 96-well plates) and incubate at RT overnight.</li>
	<li>The following day, remove the liquid and wash three times with distilled sterile water.</li>
	<li>Let the plates dry at RT overnight. The following day, add a laminin solution diluted in PBS (5 &#181;g/mL; 40 &#181;L/well for 96-well plates) and incubate for 2 h at 37 &#176;C.</li>
</ol>
6. Cell seeding
<ol>
	<li>When the oligospheres reach a diameter of 100&#8211;150 &#181;m, they are ready to be dissociated and seeded on the poly-D,L-ornithine/laminine coated plates. To do so, transfer the entire cell suspension to a 15 or 50 mL tube, and centrifuge for 5 min at 400 x g (as indicated in step 4.1)</li>
	<li>Remove the entire volume by inversion and resuspend the pellet in 180 &#181;L of fresh standard culture medium. Pipette 50 times to allow disaggregation of the spheres.</li>
	<li>Add 810 &#181;L of fresh standard culture medium and count the cells.</li>
	<li>Remove the laminin solution from the wells and plate the cells at 3,000 cell/cm2 density (100 &#181;L/well for 96-well plates).</li>
</ol>
7. OPC differentiation induction
<ol>
	<li>After 3 days, remove the entire medium and add the same volume of oligodendrocyte differentiation medium.</li>
	<li>Change half of the medium every 4 days and add fresh differentiation mix (T3/CNTF/NAC) every 2 days.</li>
</ol>
8. Induction of inflammation-mediated differentiation block
<ol>
	<li>After neurosphere dissociation and oligosphere production (section 4), add the cytokine mix to the culture medium and keep oligospheres exposed to cytokines for the whole spheres formation step. 
	NOTE: The volume depends on the number of cells, since for the spheres forming cells are seeded at 10&#8211;50 cells/&#181;L.</li>
	<li>If the medium needs to be changed, change the entire volume and add the cytokine mix once more.</li>
</ol>
9. Induction of oxygen-glucose deprivation cell death
<ol>
	<li>At -1 DIV (2 days after cell seeding in multiwell plates), remove the medium and conserve it in a new multiwell plate.</li>
	<li>Add half the volume (50 &#181;L for 96-well plates) of OGD-medium (OGD group) or fresh medium (control group). The half amount of volume is used to reduce the exchange of oxygen between the liquid and the air.</li>
	<li>Place the OGD group cultures in an airtight hypoxia chamber saturated with 95% N2 and 5% CO2. To achieve saturation of the chamber, let the gas mixture flow for 6 min at 25 l/min before closing the chamber pipes.</li>
	<li>Incubate the hypoxic chamber in the incubator for 3 h. The control group and plates containing the medium removed and conserved at step 9.1 should also be left in the incubator.</li>
	<li>Remove the glucose-free (OGD group) or the new medium (control group) and add the medium removed and conserved at step 9.1.</li>
</ol>
10. Immunocytochemistry
<ol>
	<li>At the desired time point, fix the cells with cold 4% paraformaldehyde for 20 min at RT.</li>
	<li>Wash twice with PBS (10 min of incubation for each wash at RT).</li>
	<li>Incubate 1 h at RT with the blocking solution&#160;(PBS Triton 0.3% containing 1% BSA and 1% donkey/goat normal serum).</li>
	<li>Incubate with primary antibody mix (Table 1), diluted in PBS triton 0.3%, overnight at 4 &#176;C.</li>
	<li>Wash twice with PBS (10 min of incubation for each wash at RT).</li>
	<li>Incubate with secondary antibody (Table 1) solution diluted in PBS triton 0.3% adding Hoechst 33258 for 30 min at 37 &#176;C.</li>
	<li>Wash twice with PBS (10 min of incubation for each wash at RT).</li>
</ol>
11. HCS analysis of cell viability, lineage composition, and lineage-specific cell death
NOTE: The HCS representative images and workflow are shown in Figure 2A,B.
<ol>
	<li>Select the Compartmental Analysis algorithm from the main menu of the software (HCS Studio v 6.6.0) and select Scan from the main menu Develop Assay/Scan Plate.</li>
	<li>In the iDev window, select New and then select General intensity Measurement Tool from the Develop Assay template.</li>
	<li>Click on Create on the right side of the menu, selecting the 10x objective.</li>
	<li>This will open the Configure Acquisition menu. In this window, select the following parameters: (a) number of channels: the first one for the Hoechst nuclear staining (BGRFR_386) and one for each lineage-specific marker used in the reaction (b) select software focus on channel 1 and autofocus interval as 1 (c) select the plate model from the list.</li>
	<li>From the acquisition menu, look at the quality of the staining in different wells and different fields and manually select the exposure time by selecting Fixed Exposure Time in the menu.</li>
	<li>Once the acquisitions parameters are set, select Mini Scan on the top of the menu and select ten fields per well in two wells per experimental condition. This will allow the set-up of all the analysis parameter in a subset of fields for the entire plate.</li>
	<li>When the mini scan is finished, click on the Configure Assay Parameter to configure the algorithm of the analysis.</li>
	<li>Click on Configure Groups in the right side of the window and drag-and-drop the wells of the miniscan. Click on the Add button in the Groups sub-section to configure the different groups.</li>
	<li>Follow the workflow on the left side of the window step by step to develop the whole algorithm. First select Process Image for each channel and click on Background Removal and on the desired level.</li>
	<li>First identify and select the nuclei by nuclear staining. Click on Identify Primary Object &#8211; Channel 1 to select the real nuclei and avoid analyzing artifact and debris. For this purpose, zoom in a representative picture of nuclear staining and check whether the nuclei are well surrounded by the perimeter built by the software. It is possible to change the thresholding value and to apply segmentation algorithms to better identify single nuclei.</li>
	<li>Once the nuclei are defined correctly, click on the following step: Validate Primary Object. Select Object.BorderObject.Ch1 to avoid the analysis of nuclei at the border of each field image. Select Object.Area.Ch1 and, by moving the &#8220;low&#8221; and &#8220;high&#8221; bars on the histograms, remove all the identified debris or big-objects corresponding to aggregates or artifacts.</li>
	<li>Check all the mini scan representative images of all the experimental conditions to be sure that the selected parameters fit with all of them.</li>
	<li>Click on Identify Spots for each channel corresponding to the specific lineage markers, and select the Ring values: Width = 3 and Distance = 0. This will allow the identification of the cytoplasmatic fluorescence. According to the cell density, these values can be adapted. The software will automatically avoid the overlapping between adjacent rings.</li>
	<li>Select Reference Levels in the workflow to build the analysis. The setting of the reference levels will allow the automatic counting of condensed nuclei, based on the nuclear size and nuclear staining intensity, and of specific marker-positive cells, based on the cytoplasmatic fluorescence identified by the Ring.</li>
	<li>First click on Object.Area.Ch1. In the mini scan images, select a condensed nucleus and move the &#8220;LOW&#8221; bar on the histograms in order to select as &#8220;condensed&#8221; all the nuclei under this size.</li>
	<li>Click on Object.AvgIntensity.Ch1. In the mini scan images, select a condensed nucleus and move the &#8220;HIGH&#8221; bar on the histograms in order to select as &#8220;condensed&#8221; all the nuclei above this fluorescence intensity.</li>
	<li>Click on Object.RingAvgIntensity for each channel of lineage specific markers. Select in your mini scan images a positive cell and move the &#8220;HIGH&#8221; bar on the histograms in order to select as &#8220;positive&#8221; all the cells above this fluorescence intensity.</li>
	<li>Check all the mini scan representative images of all the experimental conditions to be sure that the selected parameters fit with all of them.</li>
	<li>On the top menu, select Population Characterization and select Event Subpopulation.</li>
	<li>As Type 1 Event, select ObjectAreaCh1 on the left list, then click on the AND &#62; button and finally select ObjectAvgIntensityCh1. This will allow the identification of condensed nuclei, as a combination of low area and high intensity.</li>
	<li>In the same window, deselect all the Scan Limits.</li>
	<li>Click on Select Features to Store in the top menu, to choose the parameters to keep in the analysis.</li>
	<li>Select Well Features and move from the left list to the right only the desired parameters: (a) SelectedObjectCountPErValidField (b) %EventType1ObjectCount (c) %High_RingAvgIntensity (For each channel of the specific lineage markers). 
	NOTE: This analysis will give as readout the total number of cells, the percentage of condensed nuclei, and the percentage of lineage-specific positive cells for each analyzed marker on the total cell number. If the percentage of the different lineages is needed only on live cells, is it possible either to keep the value &#8220;High_RingAvgIntensity&#8221; for the channel (absolute number of positive cells) and recalculate the percentage on total cell numbers after the subtraction of the percentage of dead cells.
	<ol>
		<li>Alternatively, it is possible to remove the dead cells from the analysis setting the same parameters used to identify condensed nuclei (steps 11.14&#8211;11.15) on the nuclei validation (step 11.11).</li>
	</ol>
	</li>
	<li>Select Scan Plate from the main top menu and click on the plate symbol on Scan Setting sub-menu on the top section to identify the well to analyze.</li>
	<li>Write the name of the experiment and the description and once all the settings are completed, press the play symbol.</li>
</ol>

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<li>Leong, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligodendrocyte+Progenitor+Cells+%28OPCs%29+from+adult+human+brain+expressed+distinct+microRNAs+compared+to+OPCs+in+development%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Oligodendrocyte Progenitor Cells (OPCs) from adult human brain expressed distinct microRNAs compared to OPCs in development.</a> Neurology. 82, (2014).</li>
<li>Bribián, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+heterogeneity+of+mouse+and+human+brain+OPCs%3A+relevance+for+preclinical+studies+in+multiple+sclerosis%5BTitle%5D)+AND+%22Journal+of+Clinical+Medicine%22%5BJournal%5D)">Functional heterogeneity of mouse and human brain OPCs: relevance for preclinical studies in multiple sclerosis.</a> Journal of Clinical Medicine. 9, 1681(2020).</li>
<li>Jäkel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+Human+Oligodendrocyte+Heterogeneity+In+Multiple+Sclerosis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Altered Human Oligodendrocyte Heterogeneity In Multiple Sclerosis.</a> Nature. 566, 543-547 (2019).</li>
<li>Medina-Rodríguez, E. M., Arenzana, F. J., Bribián, A., de Castro, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+to+isolate+a+large+amount+of+functional+oligodendrocyte+precursor+cells+from+the+cerebral+cortex+of+adult+mice+and+humans%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Protocol to isolate a large amount of functional oligodendrocyte precursor cells from the cerebral cortex of adult mice and humans.</a> PloS One. 8, 81620(2013).</li>
<li>Baldassarro, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+death+in+pure-neuronal+and+neuron-astrocyte+mixed+primary+culture+subjected+to+oxygen-glucose+deprivation%3A+the+contribution+of+poly%28ADP-Ribose%29+polymerases+and+caspases%5BTitle%5D)+AND+%22Microchemical+Journal%22%5BJournal%5D)">Cell death in pure-neuronal and neuron-astrocyte mixed primary culture subjected to oxygen-glucose deprivation: the contribution of poly(ADP-Ribose) polymerases and caspases.</a> Microchemical Journal. 136, 215-222 (2018).</li>
</ol>

The authors have nothing to disclose.

The complex nature of myelination/remyelination processes and demyelinating events makes the development of predictive in vitro systems extremely challenging. The most widely used in vitro drug screening systems are mostly human cell lines or primary pure OL cultures, with increasing use of more complex co-cultures or organotypic systems15. Even if such systems are coupled with high content technologies, pure OL cultures remain the method of choice when developing screening platforms16.
The spontaneous mixed culture described here represents a useful in vitro system, which takes all the main variables into account: physiological T3-mediated OPC differentiation, pathological interferents with the process, other cellular components, and age-related differences. The procedure contains a number of variables deriving from the origin of the cells (age of the animal) and the spheroids formation and manipulation. In fact, a critical step is the cell density of NSCs seeding after the isolation from the tissue, because in the optimal condition a single sphere should derive from a single proliferating cell. Since we have seen that isolated NSCs tend to aggregate and that they need their own secreted paracrine factors, seeding them in a range of 10&#8211;50 cells/&#181;L, in a t25 or t75 flask, is the best compromise to avoid cell aggregation but still allowing cells to communicate by secreting factors.
The main limitations of the technique is the lacking of a functional axonal myelination and a direct interaction with neurons, since the method takes into account only the OPC differentiation until the stage of mature OLs: CNPase/MBP-double positive cells with a spider net morphology. For this purpose, primary OPCs cultured on isolated dorsal root ganglia is still the main methodology17. However, the possibility to differentiate these cells from animals at any age is a fundamental point in the translational process, since it allows the test of compounds and noxious stimuli on cells isolated from the age of interest. As described here, in fact, NSCs can be isolated from both the fetal and the adult brain. Since developmental myelination and remyelination in adulthood share the same objective, i.e., to reach the nude axon and create the myelin sheath, it was originally hypothesized that the two processes were identical in every aspect, generating the so-called recapitulation hypothesis18. However, it is now clear that the two processes cannot be considered equal and that cell-intrinsic age-related differences are present and should be taken into account when choosing the most suitable in vitro model for the experimental question19. Adult NSCs-derived OPCs, in fact, show strong differences in physiological TH-driven differentiation and vulnerability to noxious stimuli14,20 as well as primary OPCs21,22. There is also heterogeneity of OPCs and OLs population in adult tissues, of particular relevance for pathological conditions23. Protocols for primary OPCs isolation from adult tissues are available24 and should be considered when the experimental question is addressed to molecules acting on remyelination in adulthood.
The differentiation of OPCs from NSCs permits the in vitro representation of the entire differentiation process, from undifferentiated precursor to mature OL. This process resembles the in vivo condition, where TH is the main driver of the process, acting through specific nuclear receptors, and it permits experimental interference with this mechanism to mimic pathological conditions in a translational view13.
The final fundamental characteristic of the model is the constant presence of astrocytes throughout the entire culture. While this makes the culture more difficult to analyze, its complex cell composition constitutes a distinct advantage. The manner in which astrocytes contribute to the response to noxious events in mixed neuronal cultures25 is widely known, and the absence of this main component of the CNS makes the in vitro system poorly predictable and translatable. On the other hand, for this characteristic, NSC-derived cultures have the disadvantage of being less uniform than single-cell type systems, and this may lead to a biased analysis. However, the cell-based HCS technique allows an analysis of the entire culture and of all the cell populations, removing also the randomization of representative fields for analysis. Assuming that the cell culture used for the experiment is of a reliable seeding quality, the HCS will give a full picture of the experimental conditions, generating statistically robust data and a number of automatic fluorescence-based analyses.
In conclusion, the current protocol describes the procedure for the isolation and differentiation of NSC-derived OPCs from fetal and adult brain. The entire protocol takes around 30 days, depending on the age of the animals and the experimental goals. In particular, spheres formation from adult origin may take double time compared to fetal ones, at the same seeding density. The time of 15 days (from -3 to 12 DIVs) after the seeding on 2D surface for the differentiation induction is, however, a fixed time in all the conditions. The full protocol allows the study of the entire TH-mediated differentiation process in a complex cellular environment, interference through specific pathological mechanisms (i.e., inflammatory cytokines and HI) and the consequent testing of new strategies aimed at overcoming these issues. The coupling of the culture model with the HCS technique generates a robust and translatable screening platform.

In the central nervous system (CNS), myelin forming cells (oligodendrocytes, OLs) and their precursors (oligodendrocyte precursor cells, OPCs) are responsible for developmental myelination, a process which occurs during the peri- and post-natal periods, and for myelin turnover and repair (remyelination) in adulthood1. These cells are highly specialized, interacting anatomically and functionally with all the other glial and neuronal components, making them a fundamental part of CNS structure and function.
Demyelinating events are involved in different CNS injuries and diseases2, and mainly act on OPCs and OLs by way of multifactorial mechanisms, both during development and adulthood. The undifferentiated precursors are driven by differentiating factors, mainly thyroid hormone (TH), in a synchronized process3 which leads the OPC to recognize and respond to specific stimuli which induce proliferation, migration to the non-myelinated axon, and differentiation into mature OLs which in turn develop the myelin sheath4. All these processes are finely controlled and occur in a complex environment.
Due to the complex nature of myelination, remyelination and demyelination events, there is a great need for a simplified and reliable in vitro method to study the underlying mechanisms and to develop new therapeutic strategies, focusing on the main cellular player: the OPC5.
For an in vitro system to be reliable, a number of factors need to be taken into account: the complexity of the cellular environment, age-related cell-intrinsic differences, physiological TH-mediated differentiation, pathological mechanisms, and the robustness of the data6. Indeed, the unmet need in the field is a model which mimics the complexity of the in vivo condition, not successfully achieved through the use of isolated pure OPC cultures. In addition, the two main components of demyelinating events, inflammation and hypoxia/ischemia (HI), directly involve other cell components that may indirectly affect the physiological differentiation and maturation of OPCs, an aspect which cannot be studied in over-simplified in vitro models.
Starting from a highly predictive culture system, the subsequent and more general challenge is the production of robust and reliable data. In this context, cell-based high-content screening (HCS) is the most suitable technique7, since our aim is firstly to analyze the entire culture in an automatic workflow, avoiding the bias of choosing representative fields, and secondly to obtain the automatic and simultaneous generation of imaging-based high-content data8.
Given that the main need is to achieve the best balance between in vitro simplification and in vivo-mimicking complexity, here we present a highly reproducible method for obtaining OPCs derived from neural stem cells (NSCs) isolated from the fetal forebrain and the adult sub-ventricular zone (SVZ). This in vitro model encompasses the entire OPC differentiation process, from multipotent NSC to mature/myelinating OL, in a physiological TH-dependent manner. The resulting culture is a dynamically differentiating/maturating system which results in a spontaneous co-culture consisting mainly of differentiating OPCs and astrocytes, with a low percentage of neurons. This primary culture better mimics the complex in vivo environment, while its stem cell derivation allows simple manipulations to be performed to obtain the cell lineage enrichment desired.
On the contrary to other drug screening strategies using cell lines or pure cultures of primary OPCs, the method described here allows the study of the effect of pathological interferents or therapeutic molecules in a complex environment, without losing the focus on the desired cell type. The HCS workflow described permits an analysis of cell viability and lineage specification, as well as lineage-specific cell death and morphological parameters.

<table><tbody><tr><td>96-well plates - untreated</td><td>NUNC</td><td>267313</td><td></td></tr><tr><td>B27 supplement (100x)</td><td>GIBCO</td><td>17504-044</td><td></td></tr><tr><td>basic Fibroblast Growth Factor (bFGF)</td><td>GIBCO</td><td>PHG0024</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Ciliary Neurotropic Factor (CNTF)</td><td>GIBCO</td><td>PHC7015</td><td></td></tr><tr><td>DMEM w/o glucose</td><td>GIBCO</td><td>A14430-01</td><td></td></tr><tr><td>DMEM/F12 GlutaMAX</td><td>GIBCO</td><td>31331-028</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td></td></tr><tr><td>EBSS</td><td>GIBCO</td><td>14155-048</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>GIBCO</td><td>PHG6045</td><td></td></tr><tr><td>HBSS</td><td>GIBCO</td><td>14170-088</td><td></td></tr><tr><td>HEPES</td><td>GIBCO</td><td>15630-056</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H3884</td><td></td></tr><tr><td>IFN-&amp;gamma;</td><td>Origene</td><td>TP721239</td><td></td></tr><tr><td>IL-17A</td><td>Origene</td><td>TP723199</td><td></td></tr><tr><td>IL-1&amp;beta;</td><td>Origene</td><td>TP723210</td><td></td></tr><tr><td>IL-6</td><td>Origene</td><td>TP723240</td><td></td></tr><tr><td>laminin</td><td>GIBCO</td><td>23017-051</td><td></td></tr><tr><td>N-acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>N2 supplement (50x)</td><td>GIBCO</td><td>17502-048</td><td></td></tr><tr><td>Non-enzymatic dissociation buffer</td><td>GIBCO</td><td>13150-016</td><td></td></tr><tr><td>PBS</td><td>GIBCO</td><td>70011-036</td><td></td></tr><tr><td>Penicillin / Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Platelet Derived Growth Factor (PDGF-AA)</td><td>GIBCO</td><td>PHG0035</td><td></td></tr><tr><td>poly-D,L-ornitine</td><td>Sigma-Aldrich</td><td>P4957</td><td></td></tr><tr><td>TGF-&amp;beta;1</td><td>Origene</td><td>TP720760</td><td></td></tr><tr><td>TNF-&amp;alpha;</td><td>Origene</td><td>TP723451</td><td></td></tr><tr><td>Triiodothyronine</td><td>Sigma-Aldrich</td><td>T2752-1G</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T1426</td><td></td></tr></tbody></table>

high-content screening differentiation and maturation analysis of fetal and adult neural stem cell-derived oligodendrocyte precursor cell cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

The first phase of the culture may vary in duration, depending on seeding density and on whether the spheres are of fetal or adult origin. Moreover, oligospheres display a reduced population doubling compared to neurospheres (Figure 1B). Moreover, spheres production from adult tissue is slower and it may take 2&#8211;3 weeks to generate oligospheres compared to fetal that may take 1&#8211;2 weeks, depending on the seeding density.
Once seeded, the entire differentiation phase of the cultures can be monitored using lineage-specific antibodies. Since the objective of this protocol is to study the final phase of the differentiation, the culture composition at 0 DIVs is not presented. However, during the first culture phase, cells will be still nestin-positive, representing neural precursors, and the majority of cells are also NG2-positive (OPCs)11. CNPase-positive cells, corresponding to the preOL stage, will be detectable 3&#8211;6 days after T3-mediated differentiation induction, while MBP-positive cells will appear between 6 and 12 DIVs (mature OLs; see Figure 2C for the cultures composition at the end of the differentiation phase).
The HCS analysis allows the detection of each single cell in the culture through the nuclear staining and the analysis of the fluorescence intensity in the remaining channels (Figure 2A,B). The composition of the culture at the end of the differentiation phase (12 DIVs) differs depending on whether the cultures are of fetal or adult origin, with fetal cultures more responsive to T3-mediated differentiation and reaching a higher percentage of mature OLs12.
Throughout the entire culture process, around 40%&#8211;50% of the cells are astrocytes (GFAP-positive cells), while a small percentage (less than 0%&#8211;10%) are neurons (beta-III-tubulin-positive cells; Figure 2C). The culture composition may vary of a 10% between different culture preparations. Adult and fetal cultures differ for the yield of mature OLs production at the end of the differentiation phase, with fetal cells showing high percentage of mature OLs, low percentage of precursors and around 30%&#8211;40% of astrocytes. On the other hand, adult cultures present more astrocytes (around 45%&#8211;55%) and less differentiated cells after 12 DIVs of differentiation induction.
To allow the software to recognize the cells and to provide a proper unbiased analysis of the culture composition, it is important that the seeding density is correct, avoiding overlapping between adjacent cells. When NSC-derived OPCs are seeded at high density, they tend to aggregate very fast, leading to the entire surface of the well being occupied by astrocytes after a few days. Moreover, mature OLs with their characteristic spider-net shape will not be visible due to the limited space (Figure 3A,B).
The inflammation-mediated differentiation block is reproducible by this in vitro assay and generates a strong decrease in preOLs and mature OLs detected by CNPase and MBP staining in both fetal and adult cultures. An increase in the number of OPCs also occurs in adult cultures (Figure 4A,B). The cytokine mix composition was chosen from in vivo experiments in a rat model of multiple sclerosis13, and was tested as an in vitro model for the inflammation-mediated differentiation block occurring in this disease.
While fetal and adult OPCs show the same vulnerability to inflammatory cytokine exposure, only fetal-derived cultures are sensitive to OGD toxicity (Figure 5A,B), showing an increase in cell death and differentiation impairment due to their different metabolic profile14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61988/61988fig01.jpg" /> 
Figure 1: Neural stem cell-derived oligodendrocyte precursor cell culture setup and differentiation protocol.&#160;(A) Scheme of the experimental procedure. (B) Representative images of neurospheres at 2, 5, and 7 DIVs, and graph showing the population doubling of neurospheres and oligospheres. Scale bar: 100 &#181;m. (C) Representative images of seeded oligosphere-derived OPCs showing the different stages of differentiation, from nestin and NG2-positive cells at 0 DIV (neural precursor/OPCs), through CNPase-positive cells at 6 DIVs (preOLs) and CNPase/MBP double positive cells at the end of the differentiation phase (12 DIVs; mature OLs). GFAP-positive cells (astrocytes) and a small percentage of beta-III-tubulin positive cells (neurons) are present throughout the entire culture. Scale bars: 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61988/61988fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61988/61988fig02v3.jpg" /> 
Figure 2: Cell-based high-content screening analysis workflows and expected differentiation readout.&#160;(A) Representative images of HCS acquisition of an entire well (96-well plate) and an isolated single field acquired with a 10x objective of a 12 DIVs culture of NSC-derived OPCs. (B) HCS analysis workflow steps including nuclei (objects) visualization, identification, and construction of nuclei ring to identify the cytoplasmic staining and marker identification. (C) Graph showing the expected culture composition at the end of the differentiation phase (12 DIVs). Markers for OPCs (PDGF&#945;R, NG2), preOLs (CNPase, APC), mature OLs (MBP), astrocytes (GFAP) and neurons (&#946;-III-tubulin) are shown for both fetal- and adult-derived cultures. Rounded off percentages for each cell markers are included in the graph, note that this is a representative experiment and percentages may be different about 5%&#8211;10%. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61988/61988fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61988/61988fig03.jpg" /> 
Figure 3: Representative high-content screening images of a high-density culture.&#160;(A) Representative image of a well (96-well plate) image acquired by 10x objective and marked for MBP expression at the end of the differentiation phase (12 DIVs). (B) Representative extracted field image highlighting the presence of aggregated cells and overlapping nuclei. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61988/61988fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61988/61988fig04.jpg" /> 
Figure 4: Expected effect of cytokine treatment on fetal- and adult-derived OPC cultures.&#160;(A) Graph showing the percentage of variation of fetal- and adult-derived OPC cultures compared to standard cultures, including the quantification of OPCs (NG2), preOLs (CNPase) and mature OLs (MBP) at the end of the differentiation phase (12 DIVs). (B) Representative images of adult cultures at the end of the differentiation phase (12 DIVs) treated with vehicle or cytokine mix and marked for NG2 or CNPase/MBP. Scale bar: 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61988/61988fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61988/61988fig05.jpg" /> 
Figure 5: Expected effect of OGD exposure on fetal-derived OPC cultures.&#160;(A) Graph showing the percentage of condensed nuclei quantified by cell-based HCS in control (ctrl) and OGD-exposed cultures. (B) Representative images of HCS-processed objects highlighting the identified condensed nuclei (white arrows). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61988/61988fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Antibody</td>
			<td>Species</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td colspan="2">anti-&#946;-III-tubulin (R&#38;D system)</td>
			<td>mouse</td>
			<td>1:3000</td>
		</tr>
		<tr>
			<td colspan="2">anti-GFAP (Dako)</td>
			<td>rabbit</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td colspan="2">anti-NG2 (Millipore)</td>
			<td>rabbit</td>
			<td>1:350</td>
		</tr>
		<tr>
			<td colspan="2">anti-PDGF&#945;R (Santa Cruz Biotechnology)</td>
			<td>rabbit</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td colspan="2">anti-CNPase (Millipore)</td>
			<td>mouse</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td colspan="2">IgG2b anti-APC, clone CC1 (Calbiochem)</td>
			<td>mouse</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td colspan="2">Anti-MBP (Dako)</td>
			<td>rabbit</td>
			<td>1:250</td>
		</tr>
		<tr>
			<td colspan="2">Anti-nestin (Millipore)</td>
			<td>mouse</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td colspan="2">Alexa Fluor 488-conjugated anti mouse (ThermoFisher Scientific)</td>
			<td>donkey</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td colspan="2">Alexa Fluor 647-conjugated anti- mouse IgG2b (ThermoFisher Scientific)</td>
			<td>goat</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td colspan="2">Alexa 568-conjugated anti-rabbit (ThermoFisher Scientific)</td>
			<td>donkey</td>
			<td>1:500</td>
		</tr>
	</tbody>
</table>
Table 1: List of primary and secondary antibodies.

Watch this Scientific Journal Video about High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures at JoVE.com

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

high-content-screening-differentiation-maturation-analysis-fetal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.8K Views.  University of Bologna. Het protocol combineert leeftijdsspecifieke isolatie van van neurale stamcellen afgeleide spontane gemengde culturen die differentiërende oligodendrocytenvoorlopercellen en astrocyten bevatten, assays die pathologische omstandigheden nabootsen en robuuste, hooggehalte uitlezingen. Deze kenmerken maken het mogelijk om ontwikkelingsmyelinisatie en volwassen remyelinisatie differentieel te bestuderen in fysiologische en pathologische omstandigheden en in een micro-omgeving die rekening houdt me...

Video: High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Developmental Biology

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

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

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

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

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Biology

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

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

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

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

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

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

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

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

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

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Source: Assetta, B.,et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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

Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

Source: Baldassarro, V. A. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61988">High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures.</a> J. Vis. Exp. (2021)
This video demonstrates a detailed protocol for isolating the lateral ventricle walls from the adult mouse brain and performing enzymatic tissue dissociation to obtain single cells. These cells are then subjected to specific culture conditions that selectively enhance the proliferation of neural stem cells and promote their differentiation into co-cultures of oligodendrocytes and astrocytes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Solutions and reagents

	Prepare ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

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