Hello everyone,
We are trying to differentiate the KOLF-NGN2 hiPSC into neurons - Article A reference human induced pluripotent stem cell line for lar...
. The differentiation is characterized by 2 phases, a pre-differentiation time that last 3 days, and the cells are in N2/Pre differentiation media (Knockout DMEM/F12NEEA; N2 supplement (1X); 10 ng/ml NT-3; 10ng/ml BDNF; 1 mg/ml Mouse Laminin). At Dif -3 the cells are plated with 1 mg/mLROCK inhibitor(Y-27632) and 2ug/ml of fresh Doxycycline. The RI is removed from the medium at Dif -2, and a full medium change is done everyday with fresh Dox.I am struggling a lot with keeping my iN alive during this process.
I already try to troubleshooting the problem but after try different coatings, I still have the same issue.
Does someone has a similar problem or an idea of something that I can try/do to overcome it? Do you think that increasing the Dox concentration will improve to avoid neuronal death? Thanks a lot I really appreciate any suggestion, advice,
Cheers,
Rita
Dear Rita,
Most of the time poor cellular viability of hiPSC derived neurons is linked to poor cell adhesion.
What is your coating protocol ?
In our lab, we experienced major issues using matrigel when trying to differentiate neural progenitors into neurons. We switched to poly-D-Lysine (50µg/mL, 2 hours at 37°C) followed by Laminin (20µg/mL, 1h at 37°C) and the cells are now adhering properly.
Best,
Arnaud