This protocol describes an easy and cost effective method to purify stem cells from the postnatal cerebellum. These stem cells account for the late-born molecularly garbled interneurons, but they also account for postnatally-derived cerebellar astrocytes. Thus, studying these cells is clearly important for understanding the development of the cerebellum.
Using this protocol, the yield of stem cell is comparable to that of FACS-based strategy which is typically 10 times more expensive. This protocol can be generalized to extract prominin-1 expressing stem cells from other tissues such as intestine and bone marrow. Begin by placing the dish with the brain under a dissection microscope.
Use fine number five forceps to remove the meninges and large blood vessels from the cerebellum, then separate the cerebellum from the brainstem using the spatula. Transfer the cerebellum into a 15 milliliter centrifuge tube containing five milliliters of ice cold HBSS solution. Rinse it then decant the HBSS.
Repeat the wash two more times. After the last rinse, add five milliliters of papain-based tissue dissociation solution that has been pre-warmed to 37 degrees Celsius. Incubate the tissue for 15 minutes in a 37 degree Celsius water bath and slowly mix the contents of the tube by inverting it three to five times every three minutes.
Prepare a wide diameter and narrow diameter glass Pasteur pipette by fire polishing over a Bunsen burner. After the incubation, wash the tissue three times with five milliliters of HBSS solution making sure to avoid losing tissue while decanting. After the last HBSS wash, add five milliliters of DPBS with 250 microliters of DNAse to the tissue and dissociate it by gently triturating 10 to 15 times with the wide diameter Pasteur pipette.
Then use the narrow Pasteur pipette to further triturate the tissue slurry 10 times. If larger pieces of tissue remain, press them against the bottom of the tube with the pipette tip and continue pipetting until a fine suspension is achieved. Place the centrifuge tubes on ice.
Then strain the dissociated cells through a 40 micrometer cell strainer into a 50 milliliter tube. Top the filter with 10 milliliters of ice cold HBSS solution to ensure that the cells pass through the mesh. Transfer the filtered cells to a fresh 15 milliliter centrifuge tube and centrifuge the suspension at 300 times g for 10 minutes at four degrees Celsius.
Then use a vacuum aspirator to remove the supernatant completely. Resuspend the cell pellet in 160 microliters of ice cold magnetic column buffer. Add 40 microliters of anti-prominin-1 microbeads to the cell suspension.
Then incubate the tubes in a refrigerator for 15 minutes to allow the antibody to bind to the prominin-1 expressing cells. Wash the cells with one to two milliliters of column buffer X and centrifuge them at 300 times g for 10 minutes. Aspirate the supernatant and resuspend the pellet in one milliliter of column buffer X.Place the magnetic columns on the magnetic stand and rinse them once with 500 microliters of buffer X.Apply the labeled cell suspension onto the column and collect the flowthrough in fresh 15 milliliter tubes.
Wash the column three times with 500 microliters of buffer X.Then remove the columns from the magnetic field and place them in 1.5 milliliter tubes. Add one milliliter of culture medium and push the plunger into the column to flush out the cells tagged with prominin-1 beads. To passage the neurospheres, transfer them along with the culture medium into a sterile 15 milliliter centrifuge tube.
Pellet the neurospheres by centrifugation at 300 times g for five minutes and discard the supernatant. Resuspend the pellet in five milliliters of dissociation media with papain or 0.05%trypsin solution and incubate the cells at 37 degrees Celsius for 10 minutes. Centrifuge the cell suspension again.
Then resuspend the cells in five milliliters of neurosphere medium and dissociate them by slowly pipetting them up and down 10 times with a Pasteur pipette. Plate the cells in neurosphere media as described in the text protocol and enrich them with secondary neurospheres after seven to 10 days in culture as previously described. Count the cells on a hemocytometer and plate the optimal number of cells on poly-D-lysine coated plates for future experiments.
Column purified prominin-1 positive postnatal cerebellar stem cells formed neurospheres in growth factor rich neurosphere medium. These neurospheres were positive for prominin-1, their marker used for isolation and for other stem cell markers such as nestin and GFAP. Cells in the flowthrough stained negative for stem cell markers prominin-1 and nestin and positive for neuronal marker beta-III tubulin.
Upon withdrawal of growth factors and in the presence of leukemia inhibitory factor or PDGF-AA, the prominin-1 positive neurospheres differentiated into neurons, astrocytes and oligodendrocytes. When attempting this technique, it is important to make sure the neurospheres are pushed through for prominin-1 antibody and they can be passaged because normal cells also tend to form clumps which may look like neurospheres. Using this isolation method, one can characterize its derivative lineages like neurons and glia in health and disease condition.
This method is valuable and provides novel insight into role of cerebellar stem cells in cerebellar development and disease conditions like spinocerebellar ataxia and cancer.
