The overall goal of this protocol is to characterize early brain developmental processes of human neural precursor cells in a rapid and reproducible fashion, and to define the effects of important extracellular factors in regulating these processes. This method can help address important questions in the fields of neuropsychiatry and neurodevelopmental disorders such as do all affected individuals have the same cellular abnormality, or alternatively, does each display a person's specific dysfunction? The main advantage of our protocol is that it's rapid, highly-reproducible and easy to implement.
Though this method can be used to provide insight into neurodevelopmental disorders, it can also be used to study neurodegenerative and neuropsychiatric disorders such as Alzheimer's and depression. Visual demonstration of some of these methods are necessary as some techniques involve moving cells from one dish to another or require morphological analyses which are best communicated through visual demonstration. To lift the human neural precursor cells, aspirate the medium and wash the cells once with 1X phosphate buffered saline.
Aspirate the phosphate buffered saline and add 500 microliters of 1X cell detachment solution to a well with the neural precursor cells. Then, incubate the plate at 37 degrees Celsius for 10 minutes. Next, use a P1000 pipette to add 500 microliters of phosphate buffered saline maintained at room temperature to wash and remove the cells.
Then, transfer the cells in phosphate buffered saline into a 15-milliliter conical tube. Wash the well again with one milliliter of phosphate buffered saline and transfer the wash to the tube. Next, spin the cells at 300 G for five minutes to form the pellet.
Then, discard the supernatant and resuspend the pellet in one to five milliliters of pre-warmed DMEM F12 medium. Plate 100, 000 cells in triplicate wells in a 24-well plate. After 46 hours, add tritiated thymidine to the culture medium and incubate at 37 degrees Celsius for two hours.
Then, after removing the medium, add 300 microliters of pre-warmed 0.25%trypsin EDTA and incubate for 20 minutes at 37 degrees Celsius to break the cells apart and release the DNA. Then, turn the cell harvester and pump on. Place a filter paper in the cell harvester.
Keep collecting the tubes of the cell harvester in an empty blank tray. Then, press prewash to wet the filter paper. Place the collecting tubes in sample wells and start.
Use a hot light source to dry the filter paper and arrange corresponding vials on the tray. Punch out paper chads into the vials and then add two milliliters of liquid scintillation cocktail to each vial. Incubate the vials for one hour in the scintillation counter before operating the machine to obtain the counts per minute of each sample which reflects the active DNA synthesis.
Seed 500, 000 cells in a 35-millimeter culture dish. Then, agitate the dish in all directions to evenly distribute the cells. Next, incubate the cells at 37 degrees Celsius for 46 hours.
Then, add two microliters per milliliter of five millimolar EdU to the culture and incubate for two hours. Next, dissociate and pellet the cells. To the cell pellet, add three milliliters of 30%expansion medium with or without growth factors or drugs.
Plate one milliliter of the cell suspension on the pre-coated dishes and incubate the plate at 37 degrees Celsius for two hours. To study the neural precursor cell number in culture, after two days, label and prepare microcentrifuge tubes. Then, aspirate the medium from the well and add 200 microliters of 1X cell detachment solution and incubate the plate at 37 degrees Celsius for 15 minutes.
After cell detachment, add 300 microliters of 1X phosphate buffered saline to each well. Use a P1000 pipette to pipette cell solution several times and detach the cells from the plate. Then, transfer the cell solution to the prepared 1.5-milliliter tube and invert the tube two to three times.
Using a pipette, withdraw 50 microliters of cells halfway down the tube and add to trypan blue solution in a 0.5-milliliter tube. Then, count the cells using a hemocytometer. To form the neurosphere, pipette one milliliter of 100%expansion media into a 35-millimeter dish without any coating substrate.
Then, add one million neural precursor cells in each of these plates and incubate the plate at 37 degrees Celsius for 48 to 96 hours. To prepare the plate for neurosphere migration, dissolve ECM-mimic gel aliquots in six milliliters of 30%expansion medium. Add one milliliter of ECM-mimic gel with 30%expansion medium in a single well of a six-well plate.
Then, incubate the plate at 37 degrees Celsius for 30 minutes. To plate the neurospheres, use a pipette to collect the cells in the medium from the 35-millimeter dish and transfer them to a conical tube. Then, spin the tube at 100 G to pellet the neurospheres.
Then, resuspend the pellet in one to three milliliters of pre-warmed 30%expansion medium. Next, pipette 200 microliters of the resuspended neurospheres into the six-well plate containing the ECM-mimic gel with 30%expansion medium and rock the plate. Then, incubate the plate at 37 degrees Celsius for 48 hours.
Aspirate the ECM-mimic gel with 30%expansion medium and then fix the cells for 30 minutes using 4%paraformaldehyde. To analyze the neurospheres, capture images using phase-contrast settings at 10X magnification. Use the NIH ImageJ software to measure the average migration of the neurospheres.
On ImageJ, use the freehand line tool to trace the outer contour of the neurosphere. Then, use measure function to calculate the trace area, and trace the inner cell mass of the sphere. Subtract the inner cell mass from the total neurosphere area to quantify the average migration.
The immunocytochemical staining was done to study the expression of cytoskeletal proteins and transcription factors such as SOX2, PAX6 and nestin in neural precursor cells at passage three. Next, phase-contrast images were captured to identify the neurite-bearing cells. From the image, the cells with processes equal to or longer than two cell bodies are considered neurite-bearing, whereas cells with processes shorter than two cell bodies are not considered neurite-bearing.
To confirm neurosphere formation, phase-contrast images were captured which show that neurospheres collected at the 72-hour time point are between 80 micrometers and 120 micrometers in size, and therefore, can be used for migration assay. To define the proliferative capacity, neural precursor cells were grown in control or fibroblast growth factor conditions and stained for the presence of EdU. The phase-contrast fluorescent images show that cells treated with fibroblast growth factor have a higher proportion of cells engaged in S-phase as shown by EdU staining.
Next, to check for neurite outgrowth, cells were assayed in control conditions and compared to PACAP. Phase-contrast images were captured and show an increase in the number of cells with neurites under PACAP treatment. Then, to understand the neurosphere migration, the cells were treated with PACAP neuropeptides.
The phase-contrast images obtained show increased neurosphere migration in comparison to the control. Our protocol contains many techniques and assays with each requiring different amounts of time. Upon mastery, the average assay can take from two to three hours to set up and from 30 minutes to three hours to analyze.
While performing these procedures, it is critical to ensure that you are using high-quality neural precursor cells, and that these cells are completely dissociated and uniformly distributed in all conditions with the exception of the neurosphere assay. Following these procedures, other analyses can be performed including Western blotting, QRT-PCR, transcriptomics, proteomics and metabolomics in order to characterize the underlying mechanisms. Thanks for watching and good luck with your experiments.
