I derived human neural stem/progenitor cells from iPSC and have been doing RNAi screen on the NSPC. Normally they are adherent cells and form a network like structure. But when I knock down this particular gene, the cells start lifting off and forming spheres, which look like neurospheres or EBs to me. I have not done detailed characterization yet but wanted to ask if any of you have an idea what these spheres could be and what kind of experiments I could as a follow up? I am thinking of immunostaining by taking slices and also qPCR for some of the NSPC markers. Any other suggestions?
Dear Ceren,
I haven't worked with iPSC-derived NSPC, although I´ve done plenty of NSPC primary cultures from mice and rat ventricular zone.
In my experience, the primary NSPC cultures behave differently according to the culture media they are grown in. The can be amplified in B27-supplemented media as a monolayer on a Poly-L-Lysine (PL)-coated surface, or as neurospheres if no PL is used.
However, there are particular cases that render the opposite effect. For example, the NSPC suspension becomes adherent if Fetal Calf Serum is used, even on a PL-free surface. On the other hand, when planning to plate cells as a monolayer, the actual treatment may avoid cells from attaching to the PL-coated surface and you end up with viable cells growing as neurospheres. This is to say, the same cells can grow under either form according to the (1) culture media compositions, (2) the particular experimental conditions and (3) the plastic ware pre-treatment.
To complicate things a little more, this does not mean to say the NSPC may behave the same if they are grown as a monolayer network or as neurospheres, The cell-cell interactions and the exposure to nutirents and metabolic waste are significantly different in either case. Needless to say, if you´re working with this kind of system where differentiation and cell commitment is delicate, I would make sure cell proportions do not vary significantly amongst neurospheres and the monolayer for example.
I think that there is no straight forward answer to your question other than a empirical trial and error follow up on your culture system.
Did you cells form Neurospheres/EB under the control condition as well (such as with scrabled RNAi)?
Thanks, Lucas. As you say, there is probably not a straight forward way to characterize them immediately. I will be checking some of the NSPC specific markers, their expression levels, and interaction partners in addition to the morphology.
And to your question, the control (non-targeting shRNA) sample does not have the neurosphere phenotype. Untreated or control sample both have adherent, network-like structure as opposed to the KD of that particular gene.
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