I need to derive human IPSC clones from a bulk population that was stably transfected with Dox-inducible (TetOn) overexpression constructs, using the piggybac / transposase system.
I did the transfection using the Amaxa Nucleofector 4D system and I could successfully select cells for the integration of my construct (hygro). The cell line I use is the ND41865 line from Coriell
When I did serial dilutions to obtain clones from single cells, the culture looks nice and has the typical hIPSC morphology that I'm familiar with. However, upon transferring single clones into separate wells and upon expansion of the clones, the morphology is strongly altered and I can observe massive spontaneous differentiation.
Since I want to use the hIPSC clones in differentiation experiments, I want to make sure that my starting population is fine - which does not seem to be the case.
So my questions are:
- Did anyone of you generate hIPSC clones this way?
- Is the piggybac system somehow inadequate for hIPSCs (I'm coming from a mouse ESC lab, where it's a standard method)?
- May I have done something wrong upon expansion of the clones?
- May a potential leakiness of my Dox-inducible construct by the reason for spontaneous differentiation?
I would be thankful for any kind of advice or ideas that you might have!
Thanks a lot in advance!
Do you suspect that your inducible gene might cause differentiation? Is there any cause to think the gene is turned on by the presence of Tet (sometimes found in FBS - which would be present of you're culturing your hPSCs on MEFs). You could check for expression of your gene first to determine if it's leaky or not. If you think this is the problem, you might want to go with feeder-free I'm culturing my hPSCs in E8; a bit different to work with, but it definitely works. Looking for certified Tet-free FBS for your MEFs is another way to go.
Hi, I did similar work before with hESCs. Yes, if there is FBS in your culture system, it could induce tet-on to express your genes. So we use FBS-free culture media. And to inhibit the single stem cells differentiation, we use Y-27632, which is helpful to support single hPSCs survival and inhibit differentiation.
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