The overall goal of the following experiment is to generate neural stem cell cultures from different areas of the adult rodent brain. This is achieved by harvesting tissue from a region of interest to examine for the presence and potential of neural stem cells. As a second step, process the tissue into a cell suspension for plating as a monolayer culture.
Next, use defined culture conditions to selectively propagate the neural stem cells, validating their self-renewal potential. In vitro results are obtained showing that the neural stem cells are multi potent based upon their differentiation potential, and how various factors can influence their self-renewal and fate. This method can help answer key questions in the neuroscience and regenerative medicine fields, such as where endogenous neuro stem cells reside and how to manipulate them.
We first thought of this technique when we discover signals reduction pathways. That allowed us to both identify putative endogenous neuros stem cells as well as to regulate their number. So we applied this knowledge to establish robust cultures of these cells from different areas of the brain.
Study their properties and ask the question, are they indeed stem cells? Proper culture dish preparation is critical two days prior to the scheduled dissection coat the dishes with poly L ornithine at 75 micrograms per milliliter. Then incubate the plates overnight the next day, aspirate the poly L ornithine ine and wash each well twice with three milliliters of PBS for five minutes per wash.
The plates can then be stored in an incubator for up to three weeks After removing the final wash, add fiber nin diluted one to 250 in PBS by gentle pipetting. Be careful not to overly agitate the solution. Then incubate the plates overnight on the day of the dissection.
Aspirate the fibronectin and wash the dishes twice with PBS. Return the plates with the final PBS wash to the incubator and wait for the dissociated tissue. Unused plates can be stored in the incubator for up to a week, but do not let them dry out and do not agitate them.
The demonstration of this protocol is scaled to three adult rats. First, be sure to chill the brain sectioning block on ice prior to euthanizing the first rat. Then prepare a 15 milliliter conical tube on ice with five milliliters of N two medium and basic FGF.
After removing the brain from a euthanized rat, place the brain into a 10 centimeter Petri dish containing ice cold PBS. Then transfer the brain to a second dish of PBS without any residual hair or blood. Now place the brain into the chilled sectioning block and insert one new clean razor blade into the block in proximity to the region of interest from which cells will be generated.
Next, insert a second clean razor blade. Three millimeters coddle to the first razor blade. Carefully lift the blades from the block taking the section of interest with them.
Float the section off the blade by immersing it in PBS. Now harvest the area of interest using number five forceps. In this example, the section contains the anterior commissure.Sure.
Collect the section into the 15 milliliter conical tube containing five milliliters of N two medium with basic FGF. Then reduce the volume of the solution to one to two milliliters. Next, in a cell culture hood submerge a one milliliter pipette tip to the bottom of the conical and use about 20 flows of solution to mechanically dissociate the tissue.
Stop the tation when the solution becomes homogenous and allow it to sit for a few minutes so that larger unassociated tissues will settle to the bottom. Now transfer the homogenous supernatant without any tissue chunks into a conical tube containing 8.5 milliliters of supplemented and two media. Mix the tube contents and have ready a prepared six.
Well plate. Remove the PBS from the wells and then add three milliliters of cell mixture per well into three wells over the next 10 to 14 days of culturing. Replace the medium with fresh supplemented N two every other day, beginning 24 hours after plating on every other day, add a fresh volume of supplements in N two.
Medium to the medium already on the cells. Diligence to the media conditions is essential to keep the cells undifferentiated and allow for their expansion. After the first medium change, a large amount of cellular debris remained along with blood vessels.
However, over the next few days, colonies of distinct proliferating stem cells became visible. Also present were more elongated spindle like cell populations. These are not neural stem cells and were eventually overtaken as the neural stem cells proliferated.
Over the course of the next two weeks, the neural stem cells grew more densely until they reached confluence. These cultures stained positive for a number of stem cell markers, including SOX two, hesi and nest. In thus providing confidence that the cell populations that have been selected and expanded are indeed neural stem cells.
Further confirmation came after withdrawing FGF from the medium, which allowed the cells to differentiate for 10 days into neurons, astrocytes, and all the cendra cytes. To induce differentiation. Replace the supplemented N two medium with unsu supplemented N two, medium and culture the cells this way for 10 days, replacing the N two every 48 hours after 10 days aspirate the medium and fix the cells with two milliliters of 4%para formaldehyde per well for 20 minutes.
Wash the fixative out with three milliliters of PBS twice for five minutes per wash. After aspirating the second wash, add two milliliters of PBS containing 5%normal donkey serum and 0.1%tritton X 100 to perme and block the cells while the cells incubate at room temperature for 20 minutes. Prepare the primary antibody mix in PBS containing 5%NDS.
In this example, anti T UJ one antibody is applied to identify neurons while GFAP and CPA's antibodies are used to visualize astrocytes and oligodendrocytes respectively. After 20 minutes, aspirate the blocking solution and add 1.5 milliliters of the primary antibody mixture to each. Well incubate the cells.
Add room temperature for 90 minutes after 90 minutes, wash the cells with two PBS baths, followed by one application of PBS containing 5%NDS use three milliliters of solution and five minute baths. After removing the final wash, apply 1.5 milliliters of secondary antibodies in PBS containing 5%NDS to each. Well then put the plate in the dark, add room temperature and wait 40 minutes.
After 40 minutes, replace the secondary with two milliliters of a freshly prepared DPI stain and wait three minutes. After aspirating the DPI solution, wash the cells three times with three milliliters of PBS for five minutes per wash. The cells are then ready for microscopy Following this procedure.
Other methods like western blots can be performed to look at changes at protein expression and phosphorylation that result from pharmacological treatments or over the course of cell differentiation. After the development of this technique, it actually paved the way to researchers in the field of neuroscience to test new methods that they implemented in the research in several different species, including mice, rats, and even primates.
