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...

<ol>
	<li>Michalski, J. P., Kothary, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocytes+in+a+nutshell.">Oligodendrocytes in a nutshell.</a> Frontiers in Cellular Neuroscience. 9, 340 (2015).</li><li>Verden, D., Macklin, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+by+central+nervous+system+remyelination:+molecular,+cellular,+and+functional+considerations.">Neuroprotection by central nervous system remyelination: molecular, cellular, and functional considerations.</a> Journal of Neuroscience Research. 94, 1411-1420 (2016).</li><li>Raff, M. C., Lillien, L. E., Richardson, W. D., Burne, J. F., Noble, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+growth+factor+from+astrocytes+drives+the+clock+that+times+oligodendrocyte+development+in+culture.">Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture.</a> Nature. 333, 562-565 (1988).</li><li>Crawford, A. H., Chambers, C., Franklin, R. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination:+The+true+regeneration+of+the+central+nervous+system.">Remyelination: The true regeneration of the central nervous system.</a> Journal of Comparative Pathology. 149, 242-254 (2013).</li><li>Butt, A. M., Papanikolaou, M., Rivera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+oligodendroglia.">Physiology of oligodendroglia.</a> Advances in Experimental Medicine and Biology. 11175, 117-128 (2019).</li><li>Baldassarro, V. A., Giardino, L., Calz&#224;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocytes+in+a+dish+for+the+drug+discovery+pipeline:+the+risk+of+oversimplification.">Oligodendrocytes in a dish for the drug discovery pipeline: the risk of oversimplification.</a> Neural Regeneration Research. 16, 291-293 (2021).</li><li>Buchser, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+development+guidelines+for+image-based+high+content+screening,+high+content+analysis+and+high+content+imaging.">Assay development guidelines for image-based high content screening, high content analysis and high content imaging.</a> Assay Guidance Manual. 1, 1-80 (2014).</li><li>Zanella, F., Lorens, J. B., Link, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+screening:+seeing+is+believing.">High content screening: seeing is believing.</a> Trends in Biotechnology. 28, 237-245 (2010).</li><li>Ahlenius, H., Kokaia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+generation+of+neurosphere+cultures+from+embryonic+and+adult+mouse+brain.">Isolation and generation of neurosphere cultures from embryonic and adult mouse brain.</a> Methods in Molecular Biology. 633, 241-252 (2010).</li><li>Chen, 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=Isolation+and+culture+of+rat+and+mouse+Oligodendrocyte+Precursor+Cells.">Isolation and culture of rat and mouse Oligodendrocyte Precursor Cells.</a> Nature Protocols. 2 (5), 1044-1051 (2007).</li><li>Baldassarro, V. 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=The+role+of+nuclear+receptors+in+the+differentiation+of+Oligodendrocyte+Precursor+Cells+derived+from+fetal+and+adult+neural+stem+cells.">The role of nuclear receptors in the differentiation of Oligodendrocyte Precursor Cells derived from fetal and adult neural stem cells.</a> Stem Cell Research. 37, 101443 (2019).</li><li>Baldassarro, V. 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=The+role+of+nuclear+receptors+in+the+differentiation+of+Oligodendrocyte+Precursor+Cells+derived+from+fetal+and+adult+neural+stem+cells.">The role of nuclear receptors in the differentiation of Oligodendrocyte Precursor Cells derived from fetal and adult neural stem cells.</a> Stem Cell Research. 37, 101443 (2019).</li><li>Fern&#225;ndez, M., Baldassarro, V. A., Sivilia, S., Giardino, L., Calz&#224;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+severely+alters+thyroid+hormone+signaling+in+the+central+nervous+system+during+experimental+allergic+encephalomyelitis+in+rat:+direct+impact+on+OPCs+differentiation+failure.">Inflammation severely alters thyroid hormone signaling in the central nervous system during experimental allergic encephalomyelitis in rat: direct impact on OPCs differentiation failure.</a> Glia. 64, 1573-1589 (2016).</li><li>Baldassarro, V. A., Marchesini, A., Giardino, L., Calz&#224;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+glucose+deprivation+on+the+survival+of+fetal+versus+adult+neural+stem+cells-derived+Oligodendrocyte+Precursor+Cells.">Differential effects of glucose deprivation on the survival of fetal versus adult neural stem cells-derived Oligodendrocyte Precursor Cells.</a> Glia. , 23750 (2019).</li><li>Merrill, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+pharmacological+models+to+assess+demyelination+and+remyelination.">In vitro and in vivo pharmacological models to assess demyelination and remyelination.</a> Neuropsychopharmacology. 34, 55-73 (2009).</li><li>Lariosa-Willingham, K. D., 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+high+throughput+drug+screening+assay+to+identify+compounds+that+promote+oligodendrocyte+differentiation+using+acutely+dissociated+and+purified+oligodendrocyte+precursor+cells.">A high throughput drug screening assay to identify compounds that promote oligodendrocyte differentiation using acutely dissociated and purified oligodendrocyte precursor cells.</a> BMC Research Notes. 9, 419 (2016).</li><li>Dincman, T. A., Beare, J. E., Ohri, S. S., Whittemore, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+cortical+mouse+oligodendrocyte+precursor+cells.">Isolation of cortical mouse oligodendrocyte precursor cells.</a> Journal of Neuroscience Methods. 209, 219-226 (2012).</li><li>Fancy, S. P. J., Chan, J. R., Baranzini, S. E., Franklin, R. J. M., Rowitch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+regeneration:+a+recapitulation+of+development.">Myelin regeneration: a recapitulation of development.</a> Annual Review of Neuroscience. 34, 21-43 (2011).</li><li>Franklin, R. J. M., Ffrench-Constant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+CNS+Myelin+-+from+mechanisms+to+experimental+medicines.">Regenerating CNS Myelin - from mechanisms to experimental medicines.</a> Nature Reviews Neuroscience. 18, 753-769 (2017).</li><li>Baldassarro, V. A., Marchesini, A., Giardino, L., Calz&#224;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP+activity+and+inhibition+in+fetal+and+adult+Oligodendrocyte+Precursor+Cells:+effect+on+cell+survival+and+differentiation.">PARP activity and inhibition in fetal and adult Oligodendrocyte Precursor Cells: effect on cell survival and differentiation.</a> Stem Cell Research. 22, 54-60 (2017).</li><li>Leong, S. 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=Oligodendrocyte+Progenitor+Cells+(OPCs)+from+adult+human+brain+expressed+distinct+microRNAs+compared+to+OPCs+in+development.">Oligodendrocyte Progenitor Cells (OPCs) from adult human brain expressed distinct microRNAs compared to OPCs in development.</a> Neurology. 82, (2014).</li><li>Bribi&#225;n, 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=Functional+heterogeneity+of+mouse+and+human+brain+OPCs:+relevance+for+preclinical+studies+in+multiple+sclerosis.">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>J&#228;kel, 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=Altered+Human+Oligodendrocyte+Heterogeneity+In+Multiple+Sclerosis.">Altered Human Oligodendrocyte Heterogeneity In Multiple Sclerosis.</a> Nature. 566, 543-547 (2019).</li><li>Medina-Rodr&#237;guez, E. M., Arenzana, F. J., Bribi&#225;n, A., de Castro, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+to+isolate+a+large+amount+of+functional+oligodendrocyte+precursor+cells+from+the+cerebral+cortex+of+adult+mice+and+humans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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 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 platforms<sup class="xr...

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-&#947;</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&#946;</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-&#946;1</td>
			<td>Origene</td>
			<td>TP720760</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-&#945;</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 different...

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 ...

The protocol combines age-specific isolation of neural stem cell-derived spontaneous mixed cultures containing differentiating oligodendrocyte precursor cells and astrocytes, assays mimicking pathological conditions, and robust, high-content readouts. These characteristics make it possible to differentially study developmental myelination and adult remyelination in physiological and pathological conditions and in a microenvironment that takes into consideration the contribution of astrocytes. For neural stem cell isolation from embryonic day 13.5 to 14.5 fetal forebrains, place the heads of the embryos in a clean Petri dish with ice cold PBS and use forceps to remove the skin from the skull under magnification.Once the brain is visible and cleared of skin, use forceps to squeeze the brain out of the skull and remove the cerebellum. Use the forceps to remove the meninges and place the isolated tissue in the non-enzymatic dissociation buffer. When all of the brain tissue has been isolated, pull two to three samples in 150 microliters of non-enzymatic dissociation buffer per 1.5 milliliter tube, and incubate the tubes for 15 minutes at 37 degrees Celsius with continuous shaking.At the end of the incubation, add 850 microliters of standard medium to each tube and pipette to mix until the suspension is free of clumps. If non-dissociated tissue is still visible, allow the samples to rest for two minutes to allow the tissues to settle to the bottom of the tubes. When the dissociation is complete, plate cells at a density of 10 to 50 cells per microliter in 10 to 30 milliliters of neurosphere medium concentration in a T25 or T45 flask, and place the flask in the cell culture incubator in a vertical position to prevent cell attachment.from 2.5-month-old adult mice, place brains from four to five adult mice into a 50-milliliter tube of ice cold HBSS and place one brain, ventral side down, in the rostral-caudal direction on a sterile aluminum foil-covered flask filled with minus 20 degrees Celsius overnight-cold water. Use a razor blade to remove the olfactory bulbs and to cut two to three one-millimeter-thick coronal slices from the cortex to the optical chiasma. Place the slices on the cold surface in a ventro-dorsal position, and identify the corpus callosum and the two lateral ventricles.Using magnification, isolate the walls of the lateral ventricles, taking care not to include pieces of the corpus callosum, and place the isolated tissue in five to 10 milliliters of enzymatic dissociation buffer for a 15 minute incubation at 37 degrees Celsius. At the end of the incubation, pipette the tissues at least 50 times before incubating the samples at 37 degrees Celsius for an additional 10 minutes. At the end of the incubation, neutralize the trypsin with five milliliters of standard culture medium and filter the tissue suspension through a 70-micron filter.Centrifuge the filtered solution for five minutes at 400 times g, and re-suspend the pellet in sucrose solution for a second centrifugation. At the end of the centrifugation, re-suspend the pellet in bovine serum albumin washing solution for another centrifugation, then re-suspend the pellet in standard culture medium for counting and plate the cells in an upright T-25 or T-45 flask. For primary neurosphere differentiation, add basic fibroblast and endothelial growth factors to the neural stem cell culture every two days.When the neurospheres reach a 100-to 500-micron diameter, passage the cells by mechanical dissociation according to standard protocols. For oligosphere differentiation, treat the cells with basic fibroblast growth factor in platelet-derived factor-AA every two days. When the oligospheres reach a 100-to 150-micron diameter, dissociate the spheres by mechanical dissociation according to standard protocols and seed the cell suspension at a 3000-cell per square centimeter density onto poly-DL-ornithine laminin coated plates.After three days, replace the supernatant of each culture with the same volume of oligodendrocyte differentiation medium. To perform an inflammation-mediated differentiation block induction, after neurospheres dissociation and during oligosphere production, add the cytokine mix of interest to the culture medium. To induce oxygen-glucose deprivation cell death, two days after seeding into the multi-well plates, transfer the supernatant from each culture into individual wells of a multi-well plate.Add half the volume of OGD medium to the wells, and transfer the cultures to an airtight hypoxia chamber saturated with 95%nitrogen and 5%carbon dioxide. Place the chamber in the cell culture incubator. After three hours, replace the medium in the chambers with the medium set aside from the multi-well culture plate.After checking the quality of the cell staining, in the Acquisition menu of the high-content screening software, select Fixed exposure time and Mini scan, and select 10 fields per well and two wells per experimental condition to allow the analysis parameters in the subset of fields to be set for the entire plate. Follow the workflow step-by-step to develop the whole algorithm. Select Process image for each channel and click Background removal to select the desired level of signal.To identify and select the nuclei by nuclear staining, click Identify primary object, adjust the threshold to identify single nuclei, and click on Validate primary objects to set the thresholds based on object areas and position. Click Identify spots for each channel corresponding to the specific lineage markers, and set the ring value width to three and the distance to zero to allow identification of the cytoplasmatic fluorescence. Select Reference levels in the workflow to build the analysis and to allow automatic counting of the condensed nuclei based on the nuclear size and nuclear staining intensity and of the specific marker-positive cells based on the cytoplasmatic fluorescence identified by the ring, then click Play.During the first phase of the culture, the neural stem cells maintain their nestin expression. The majority of cells are also NG2-positive. CNPase-positive cells corresponding to the pre-oligodendrocyte stage are detectable three to six days after T3-mediated differentiation induction, while mature MBP-positive oligodendrocytes appear in a significant percentage after six to 12 days in vitro.High-content screening analysis allows the detection of single cells within the culture through nuclear staining and fluorescence intensity analysis. The composition of the culture at the end of the differentiation phase 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 oligodendrocytes. Note that when neural stem cell-derived oligodendrocyte precursor cells are seeded at a high density, they aggregate, and replicating astrocytes rapidly produce a confluent cell layer, preventing observation of the characteristic spider net shape of mature oligodendrocytes.Inflammation-mediated, differentiation blocking induces a strong decrease in pre-and mature oligodendrocytes, as detected by CNPase and MBP staining in both fetal and adult cultures. An increase in the number of oligodendrocyte precursor cells also occurs in cytokine-treated adult cultures. While fetal and adult oligodendrocyte precursor cells show the same to inflammatory cytokine exposure, only fetal-derived cultures are sensitive to oxygen-glucose deprivation toxicity.The method can be implemented with any kind of oligodendrocyte precursor cells, differentiation and maturation interfering process, to test new strategies aiming to fight diseases affecting myelination or remyelination

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

Research

JoVE Journal

Neuroscience

2.7K ビュー.  University of Bologna. このプロトコルは、分化オリゴデンドロサイト前駆細胞と星状細胞を含む神経幹細胞由来の自発的混合培養物の年齢特異的分離、病理学的状態を模倣するアッセイ、および堅牢で高含有量の読み出しを組み合わせたものです。これらの特徴は、生理学的および病理学的状態および星状細胞の寄与を考慮した微小環境における発達髄鞘化および成人再髄形成を差異的に研?...

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