使用克隆测定神经干细胞的计数。

The overall goal of this procedure is to enumerate neural stem cells frequency in a given population using neurosphere formation and differentiation under clonal conditions. This method has key utilities in the field of neural stem cells such as evaluation of candidate neural stem cell markers, purification of neural stem cells and distinguishing neural stem cells from neural progenitors. The advantages of this method are that it takes 13 days to perform which is shorter than current methods to numerate neural stem cell frequency and is performed under clonal conditions.

To begin this procedure, on day one, prepare NSC growth medium by combining DMEM F12 medium with B27, EGF, BFGF, and penicillin streptomycin. Next dissociate E14.5 mouse neurospheres in one milliliter of NSC growth medium and one milliliter of 0.05 normal Sodium Hydroxide for seven minutes at room temperature. Pipette the cells up and down occasionally to keep them in suspension, then, add one milliliter of 0.05 normal hydrochloric acid to neutralize the alkali.

After that, perform a cell count using trypan-blue staining and a hemocytometer. Following this seed single E14.5 mouse neurosphere cells at a density of 50 cells per milliliter or below in 100 microliters of NSC growth medium in a 96 well dish. Incubate the cells at 37 degrees Celsius, 5%CO2 for one week to generate clonal neurospheres.

To culture neurospheres for the conditioned medium, on day three dissociate E14.5 mouse neurospheres and seed the single cells at a density of 20, 000 cells per millilter in 10 milliliters of NSC growth medium in a 10 centimeter culture dish. Then incubate the cells at 37 degrees Celsius, 5%CO2 for five days to generate bulk cultured neurospheres. On day eight prepare the coating solution by mixing 700 microliters of 0.01%poly-L-lysine, 70 microliters of laminin, add one milligram per millilter and 6, 230 microliters of PBS.

Next, coat a 10 centimeter culture dish with seven milliliters of poly-L-lysine laminin solution for two hours at 37 degrees Celsius. Using a 10 milliliter serological pipette transfer all the bulk cultured neurospheres to a 15 millimeter conical centrifuge tube. Centrifuge the neurospheres at 171 times g for one minute at room temperature, and remove the supernascence completely.

After that, prepare NSC differentiation medium by combining DMEM F12 medium with B27 and FBS. Add 10 milliliters of NSC differentiation medium to the neurospheres and pipette up and down a few times to resuspend the neurospheres. Then aspirate the poly-L-lysine laminin from the 10 centimeter culture dish and wash once with 10 milliliters of PBS.

Transfer all the neurospheres to the poly-L-lysine laminin coated 10 centimeter culture dish, incubate them over night at 37 degrees Celsius, 5%CO2 for the neurospheres to adhere to the dish. On day eight count the number of neurospheres formed in each well under a microscope then coat each well of a 50 well chambered cover slip with 10 microliters of poly-L-lysine laminin coating solution for two hours at 37 degrees Celsius. Next, prepare three 50 milliliter conical centrifuge tubes containing 30 milliliters of PBS each.

Using sterile forceps remove the coated 50 well chambered cover slip, dip the chambered cover slip into the first tube of PBS, followed by the second, and then the third tube of PBS to wash off the coating solution. Afterward, aspirate any remaining liquid from the wells of the chambered cover slip. Gently transfer all the medium and neurospheres from each well of the 96 well dish to a single 10 centimeter culture dish.

Ensure that only a single neurosphere is picked each time by setting the pipette column to two microliters under the microscope pick a sample neurosphere using a P10 micro pipette fitted with a 10 microliter pipette tip. Neurospheres may be picked under the microscope, placed in a laminar flow hood, or on a bench top. Then transfer the neurosphere on to the center of a well of the coated 50 well chambered cover slip.

Repeat the procedures until each well of the chambered cover slip contains a neurosphere. Subsequently add 10 microliters of NSC differentiation medium to each well of the chambered cover slip. Place the chambered cover slip in 10 centimeter culture dish.

Up to two chambered cover slips can be placed in a 10 centimeter dish. Pipette PBS along the edges of the dish to prevent drying of the NSC differentiation medium. Incubate the chambered cover slip over night at 37 degrees Celsius, 5%CO2.

On day nine, transfer the conditioned medium from the bulk cultured neurospheres to a 15 milliliter conical centrifuge tube. Using a 20 milliliter sterile syringe pass the conditioned medium through a 0.2 micrometer filter to remove any cells or debris. Then, transfer the filtered conditioned medium back to the 10 centimeter culture dish.

After that using sterile forceps gently place the chambered cover slip onto the conditioned medium in an inverted manner such that the sample neurospheres are facing downwards. Incubate the chambered cover slip for three days at 37 degrees Celsius, 5%CO2. On day 12 gently remove the chambered cover slip from the 10 centimeter culture dish using sterile forceps.

Gently dip the cover slip into PBS to wash off the conditioned medium. Then, peel off the silicon gasket from the cover slip, perform this gently and slowly to avoid breaking the cover slip. Take note of the side where the differentiated neurospheres are adhered.

After that, pipette one millimeter of 4%paraformaldehyde on to a piece of paraffin film cut to the size of the cover slip. Gently place the cover slip on the paraffin film with the neurospheres facing downwards in contact with the paraformaldehyde and fix the neurospheres for 20 minutes at room temperature. Here, single neurospheres were picked and differentiated in a 50 well chambered cover slip for three days.

This is a representative image of a tripotent neurosphere stained with dapi and a immunostained for GFAP, TUJ1, and O4.This image shows the overlay of dapi GFAP, TUJ1 and O4.This protocol is used to assess the ability of LeX, a known NSC marker, to enrich for NSC's. This graph shows the percentage of unipotent, bipotent, and tripotent neurospheres generated by LeX positive and LeX negative cells. And LeX positive cells generate significantly more tripotent neurospheres than LeX negative.

This table shows that the NSC frequency of LeX positive and LeX negative cells was calculated as the product of NFU and the percentage of tripotent neurospheres. After watching this video, you should have a good understanding of how neurospheres can be cultured and differentiated under clonal conditions to enumerate neural stem cell frequency. While performing this method it's important to use freshly prepared growth and differentiation media and freshly prepared coating solution to ensure optimal growth and differentiation conditions for the neurospheres.

Once mastered this method can be performed in 13 days, a shorter time compared to the current methods to enumerate neural stem cell frequency. This method could be used along with current methods such as the neural colony forming cell assay to enrich for newer stem cells and identify a definitive neural stem cell marker.