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
