Neural Stem Cells

The ins and outs of Neurospheres

The isolation of neurogenic cells from mammalian brain was reported about a decade ago (for review see 1). Subsequently, a population of multipotent, self-renewing cells was identified in the adult CNS (2-3). Today, the neurosphere assay is the most common way of isolating and expanding neural stem cells in vitro (4). So what are "neurospheres"? Neural stem cells can be isolated by taking brain tissue from, for example, embryonic day 14.5 cortex, followed by mechanical dissociation and then plating on culture dishes containing serum-free defined media with EGF. After about 6 days free floating colonies of nestin positive cells can be seen, with each colony containing approximately 200-500 cells (Figure 1). They can be expanded either by cutting the neurospheres into 4-8 pieces or by dissociation into single cells followed by reculturing.

If neurospheres are plated on laimnin, the EGF withdrawn and serum added, they can be seen to differentiate into the three main cell types of the brain, astrocytes, oligodendrocytes and neurons. In particular cells at the periphery of the neurosphere begin to migrate and differentiate while cells at the center remain undifferentiated. By selecting the plating matrix and growth factors the proportion of the three cell types can be controlled (5). For example, NB3 has recently been shown the promote oligodendrocyte formation from mouse brain neurosperes (6).

Neurospheres are formed because daughter neural stem cells remain attached to their mothers through many rounds of cell division. So are all the cells in a neurosphere the same? This is an important question that has only been partially answered. Neurospheres are comprised of a heterogenous mix of neural stem cells, neural progenitors, differentiated cells and extracellular matrix proteins (7). Clonal analysis where single cells are isolated and characterised has to be carried out to investigate the exact make-up of neurospheres. Culturing neurospheres from single isolated cells is one way of doing clonal analysis, however, growing cells at very low density may lead to selection of unique populations that are not representative. We will have to identify novel ways of following neural stem cell lineages. Nevertheless, it is clear that the neurosphere assay is a very powerful way to propagate neural stem cells and model neurodevelopment.

Looking to the future, it will be very important to understand the relationship between the cells of the colony and exactly how many different cell types there are in any one particular neurosphere. The composition of neurospheres isolated from different regions of the brain or under different growth conditions will also be important to establish.

References

1. Nature Review Neurosciences 1, (2000) 67-73.
2. Proc. Natln. Acad. Sci. USA 89, (1992) 8591-8595.
3. Science 255, (1992) 1707-1710.
4. Nature Metods 2, (2005) 333-336.
5. Nature Biotechnology19, (2001) 475-479.
6. J. Biol. Chem: 279, (2004) 25858-25865.
7. Journal of Neuroscience Research 78, 761-768 (2004).