We have observed several iPSC clones with chromosomal abnormalities grown in E8/Vitronectin and poor neuronal differentiation ability and wonder if this is purely co-incidence or a culture effect.
Hi,
We have noted that the high cntent of FGF2 present in Essential 8 can indeed be inhibitory to differentiation. Seeding cells in a low fgf containing media (like Nutristem) can overcome this. Alternatively, we have tried seeding in E8 but then adding PD03 for one day and this also helped our protocol. I'm not sure about the effects of vitronectin versus matrigel since we are using Laminin 521. In terms of chromosomal abnormalities, do you directly compare karyotpyes (or SNP data) of cultures of E8/Vitronectin and mTESR/Matrigel at the same passage and find a difference? Abnormalities can increase with passage for hEPCs. See attached papers relating to fgf signalling and self-renewal/differentiation
I have not looked at chromosomal abnormalities per se but do find that there is more nucleic acid damage in general in HPSCs cultured in E8 and mTeSR when compared to KSR containing media.
Please have a look at the following link and do let me know what your thoughts are:
http://biorxiv.org/content/early/2016/08/05/067868
Regards,
Megha
For differentiation you should be switching to E6 media (without TGF beta 1 and FGF2).
Hi experts,
Does anyone have experience in iPSC maintained in mTeSR plus, subsequently form embryoid bodies in mTeSR plus with ROCKi for 24 hours, then differentiate using mTeSR-E6 containing inhibitors/growth factors for neuronal organoids differentiation?
Thank you in advance.
Jingzhang
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