Right now, I grow them to high cell densities, but eventually my experiment demands that I be able to make clonal selections of a few of them. Is there any supplement or growth factor that I can add to the media to prevent their differentiation, even when there are only a few cells in my well? right now I am using FGF2 and laminin in N2B27 medium.
Neural precursors will differentiate with extended time in culture (days in vitro), and do not exist as a un-differentiating population in vivo (except adult NSCs). If you take precursors from a late culture (extended expansion) they are likely to be glio-genic and produce astrocytes instead of neurons.
However, there is a way round this - what you can do is rapidly expand up your neural progenitors post-neuralisation (formation of neural rosettes in dense monolayer culture) using 20ng/mL FGF2 and EGF. Once you have a large enough population you can bank them by freezing them down in aliquots and storing in liquid nitrogen. Then when you need to perform an experiment, thaw up enough to get a starter well and expand once again using 20ng/mL FGF2 and EGF. Effectively these neural precursors should all be from the same initial population and therefore the same batch. Also adding 200uM 2AAP (ascorbic acid) has been shown to increase progenitor proliferation.
One warning note is you may wish to limit treatment with FGF2 and EGF to 2 days at a time, as extended FGF2 in culture has been shown to ventralise neural precursors to GABAergic neuronal fate. Unfortunately you will never get absolutely no differentiation - but passaging the wells prior to your selections may kill off any differentiated cells leaving you with mostly progenitors.
When neural precursors are at a low density they are also more likely to differentiate, as they have lost inhibitory cues from adjacent progenitor cells.
Hope this helps,
James.
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