In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum-free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments. Supplements at a 9:1 ratio, respectively. 2. The medium is warmed up in a 37°C water bath. 3. Cold HEPES-buffered minimum essential medium (HEM) with high concentration of antibiotics (10%) is prepared for dissection and washing purpose. Alternatively, NSC basal medium with antibiotics supplementation may also be used for this purpose. 4. 25-30 mL of cold HEM containing antibiotics is dispensed into sterile 50 mL tubes for collection of the embryos. 5. Several 10cm plastic Petri dishes are needed to hold the embryos and brains during dissection and also to hold dissected tissue. 6. The surgical tools, needed to remove the embryos (large scissors, small pointed scissors, large forceps, small curved forceps) or for embryonic brain dissection (small forceps, curved fine forceps, 45°angled fine forceps, and small scissors) are sterilized using glass bead sterilizer at 250°C or other available autoclave methods. 7. Dissection microscope is wiped with 70% alcohol and set up inside a laminar flow or PC2 hood.1. A time mated pregnant mouse is anesthetized on day 14 th of gestation according to one's institutional approved animal protocol. 2. Cervical dislocation is performed to make sure the animal does not suffer pain and distress. 3. The anesthetized mouse is laid on its back on an absorbent tissue paper, and the abdomen is rinsed with 70% ethanol to sterilize the area. 4. The skin over the abdomen is grasped using a large forceps, and then the skin and the underlying fascia is cut with large scissors to expose the abdominal cavity and the uterine horns. 5. The uteri are removed with small f...

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments. Supplements at a 9:1 ratio, respectively. NSC medium can also be made in laboratory based on NSC growth medium composition published in the literature 1 . If your lab does not have much experience with making medium, we strongly recommend to use commercially available medium that has been quality controled for NSCs growth prior to being sold (which is the case for NeuroCult, Stem Cell Technolgies). 2. The medium is warmed up in a 37°C water bath. 3. Cold HEPES-buffered minimum essential medium (HEM) with high concentration of antibiotics (10%) is prepared for dissection and washing purpose. Alternatively, NSC basal medium with antibiotics supplementation may also be used for this purpose. 4. 30-40 ml of cold HEM containing antibiotics is dispensed into sterile 50 ml tubes for brain collection. 5. Several 10cm plastic Petri dishes are needed to hold the brains during dissection and one 10cm sterile glass Petri dish to hold dissected tissue. 6. The surgical tools needed to remove the brain (large scissors, small pointed scissors, large forceps, small curved forceps, and a small spatula) or for tissue dissection (small forceps, curved fine forceps, and scalpel) are sterilized using glass bead sterilizer at 250°C or other available autoclave methods. 7. Dissection microscope is wiped with 70% alcohol and set up inside the PC2 hood.1. 2-4 ...

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Repetitive transcranial magnetic stimulation (rTMS) is a new method for treating many neurological conditions; however, the exact therapeutic mechanisms behind rTMS-induced plasticity are still unknown. Neural stem and progenitor cells (NS/PCs) are active players in brain regeneration and plasticity but their behavior in the context of rTMS therapy needs further elucidation. We aimed to evaluate the effects of rTMS on proliferation and differentiation of NS/PCs in the subventricular zone (SVZ) of adult mouse brain. Adult male mice (n=30) were divided into rTMS (1-Hz and 30-Hz) and sham groups and treated for 7 or 14 consecutive days. Harvested NS/PCs from the SVZ were cultured in the neurosphere assay for 8 days and the number and size of the resulting neurospheres as well as their in vitro differentiation capacity were evaluated. After one week of rTMS treatment at 1-Hz and 30-Hz compared with sham stimulation, the mean neurosphere forming frequency per brain was not different while this measure significantly increased after two weeks (P<0.05). The mean neurosphere diameter in 1-Hz treatment paradigm was significantly larger compared with sham stimulation at both 1 and 2 weeks. In contrast, 30-Hz treatment paradigm resulted in significantly larger neurospheres only after 2 weeks. Importantly, rTMS treatment at both frequencies increased neuronal differentiation of the harvested NS/PCs. Furthermore, one week in vitro rTMS treatment of NS/PCs with both 1-Hz and 30-Hz increased NS/PCs proliferation and neuronal differentiation. It is concluded that both 1-Hz and 30-Hz rTMS treatment increase NS/PCs proliferation and neuronal differentiation.

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