Isolation and Expansion of Neurospheres from Postnatal (P1−3) Mouse Neurogenic Niches

The neurosphere assay is useful in vitro technique for studying the inherent properties of neural stem/progenitor cells including proliferation, self-renewal and multipotency in both physiological and pathological context. Due to its three dimensional structure, this technique is a powerful tools for studying adult neurogenesis. It is also useful for culturing and obtaining higher yields of neural stem/progenitor cells.

This neurosphere assay can be applied to the study of brain disorders such as Alzheimer's and Parkinson's diseases or multiple sclerosis. Neurospheres can also be obtained from other brain regions or systems, such as adipose tissue or the gut. Demonstrating the procedure with Filipa Ribeiro will be Rita Soares, a PhD student in my group.

To harvest postnatal day one to three mouse brains, hold the ventral part of the body at the base of the head and use small pointed scissors to make a midline incision in the skin over the entire length of the head to expose the skull. Make a longitudinal incision at the base of the skull and continue cutting along the sagittal suture at a shallow an angle as possible to avoid damaging the brain structures. Use curved forceps to peel the skull to the sides to expose the brain and slide a small spatula under the brain to cut the cranial nerves and blood vessels that are connected to the base of the brain.

Place the brain into a Petri dish of cold HBSS supplemented with antibiotics and place the dish under a dissecting microscope at low magnification. Position the brain on its dorsal surface. While holding the brain by the cerebellum, use fine forceps to remove meninges from the ventral side of the brain and the olfactory bulbs.

Rotate the brain onto the ventral aspect and peel off the rest of the meninges. Then use the forceps to remove the cerebellum and use curved pointed forceps to transfer the brain onto a piece of filter paper with an 11 micrometer pore on top of a tissue chopper. For SVZ and DG microdissection, begin by chopping the brain into 450 micrometer coronal sections.

Use a wet lamina to collect the sectioned brain into a new Petri dish filled with cold antibiotic supplemented HBSS under a dissecting microscope and use forceps to separate coronal slices in an anterior-to-posterior fashion until slices with the lateral ventricles are reached. Use fine forceps to cut the thin layer of the tissues surrounding the lateral wall of the ventricles, excluding the striatal parenchyma and the corpus callosum and place one tip of the forceps immediately under the corpus callosum and the other into the tissue immediate adjacent to the ventral area of the lateral ventricle to isolate the SVZ. Cut a small line of tissues surrounding the lateral ventricle and collect the dissected tissue into a sample tube containing supplemented HBSS solution.

When all the slices have been microdissected with forceps and hippocampal formation has been reached, discard the first slice with hippocampus in which the DG is still unrecognizable. To remove the DG, first isolate the hippocampus from the slices and refocus the microscope to visualize the borders around DG.To harvest the DG, use forceps to make a cut between the DG and CA1 region followed by a vertical cut between the DG and CA3 region. Then remove the fimbria and any adjacent tissue and place the harvested tissue into separate tube of antibiotic supplemented HBSS solution.

To dissociate the SVZ and DG samples, add Trypsin-EDTA 0.05%to a final concentration of 5-10%of Trypsin-EDTA 0.05%in HBSS to each tube for an approximately 50 minute incubation at 37 degree Celsius. The whole use of trypsin or too long incubation time can lead to increased cell death, negatively impacting cell growth. When the tissue has clumped together, replace the enzyme solution for four consecutive washes with one milliliter of fresh supplemented HBSS per wash.

After the last wash, resuspend the digested tissues in one milliliter of serum-free medium supplemented with 10 nanograms per milliliter of epidermal growth factor and five nanograms per milliliter of basic fibroblast growth factor per tube. Then mechanically dissociate the tissues with gentle pipetting seven to 10 times with a P1000 pipet until a homogenous cell solution has been obtained. To determine the density of the dissociated DG or SVG cell, count the cells in each suspension on a hematocytometer.

To expand the cells into neurospheres, dilute the individual cell populations at a two times ten to the fourth cells per milliliter density in serum-free medium supplemented with growth factors and seed five milliliters of the cell suspension into uncoated 60 millimeter diameter Petri dishes. Then incubate the SVZ cells for six to eight days and the DG cells for 10 to 12 days at 37 degree Celsius for primary neurosphere formation. When the majority of the neurospheres have 150 to 200 micrometer diameter, harvest the cell suspension from the cultures and collect the neurospheres by centrifugation.

Resuspend the neurosphere pallet with a mouse chemical dissociation kit according to the manufacturer's instructions before collecting the cells with another centrifugation. Replace the supernatant with one milliliter of serum-free medium supplemented with growth factors and gently try to rate the pellet about 10 times. Count the dissociated neuro cells as demonstrated and reseed the cells at a two times 10 to the fourth cells per milliliter density in serum-free medium supplemented with growth factors into new 60 milliliter Petri dishes.

Then return the cell to the cell culture incubator for additional six to eight or 10 to 12 days as appropriate to obtain secondary neurospheres. SVG and DG neurospheres obtained by using the neurosphere assay are composed of undifferentiated Sox2 positive, nestin positive cells. Notably, SVG derived neurospheres have larger dimensions than their DG counterparts.

Importantly, under differentiation conditions, SVG and DG-derived neural stem/progenitor cells migrate out of the neurospheres, forming a pseudomonolayer of cells. In both neurogenic regions, it is possible to observe the presence of Sox2 double positive, nestin double positive, doublecortin double negative cell pairs corresponding to symmetrical divisions indicative of self-renewal. Cell pairs in which one cell of Sox2 positive, nestin positive, and doublecortin negative, and the other cell Sox2 negative, nestin negative, and doublecortin positive demonstrating asymmetrical divisions.

And Sox2 double negative, nestin double negative, and doublecortin double positive cell pairs corresponding to symmetrical divisions indicative of differentiation. Overall, passaging changes the cell death rate at the second day in vitro. Neuritogenesis can be evaluated in neurons obtained from the differentiation of SVG and DG neural stem/progenitor cells at the beginning of differentiation.

As observed, the length and ramification of the neurites increases with differentiation. A high percentage of proliferative cells is observed in SVG compared to DG.Although the percentage of proliferating progenitors that differentiate into mature neurons is similar in both neurogenic niches. Both cell types are also able to differentiate into immature neurons, mature neutrons, oligodendrocytes precursor cells, mature oligodendrocytes, and astrocytes.

After obtaining the neurospheres, several methods including immunocytochemistry, calcium imaging, Western blots and RT-PCR can be performed to study the stemness and the multipotency of the neural stem/progenitor cells. The neurosphere assay can be applied to genetic and disease models to further evaluate the molecular and cellular processes involve in both the proliferation and differentiation of neural stem/progenitor cells.