Hi, I have been trying to induce neural induction in my mESC lines by using -DMEM-F12 + N2+B27+NEAA+ bFGF. Although I started getting rosette formation from day5/6 onward, and mature neural marker expression from day7 onward, I also had lots of other lineage (mesodermal/ endodermal) contamination. I performed this NI for a period of 14 days, changing media everyday.

To counter the other lineage contamination, I started with a different protocol using small molecule inhibitors using the following media cocktail- Neurobasal media+DMEM-F12+NEAA+Glutamax+beta-mercaptoethanol+insulin+ B27+N2+bFGF+SB+LDN. Although this protocol has worked well in the hands of another researcher using mESC HM1, it is not working at all with my lab's established mESC lines. Even until the day 8 of N.induction, I can't morphologically see any neural lineage appearance. I just came across this publication: http://www.sciencedirect.com/science/article/pii/S0898656814001971 which discusses the role of SB in inhibiting differentiation and maintaining pluripotency of mESCs. 

I am quite confused at this point, and would highly appreciate if someone could shed some light on these issues I am facing. I have cells in culture in both conditions right now, I am tempted to take out SB in some wells and see the effect, but it would be great to get some advice before I go manipulating the protocols.

Many thanks.

Regards,

Pooja.

More Pooja Khurana's questions See All
Similar questions and discussions