iPS induction difficulty from MEF
Posted 06 November 2010 - 10:58 AM
Our lab is not really stem cell lab, so no one did this before. But for the new grant, I started to do this iPS project.
Anyway, what I did so far is
Culture of irradiated MEF (purchased) as a feeder layer.
viral transfection (lenti) of Yamanaka factors to the target cell (primary MEF (P1) that I isolated)
I culture this transfected cell on top of feeder (the number of cell seeded is according to the Nature protocol), and induced reprogramming by adding doxycycline in the induction medium.
My induction medium is DMEM + ES cell qualified FBS + NEAA + sodium pyrubate + L-glutamine + P/S + BME + Lif + Doxcycline
its been about two weeks since I started to do this induction and I am changing the medium every other day,
but now this cell culture looks like just over confluent culture with many round cell attach to the top of the fibroblast layer.
Is this how it suppose to look like??
or Am I doing something wrong??
I can find picture of iPS colony but cannot find any pictures of how it looks like from MEF to iPS colony intermediate, so I am bit worried about I might be doing something wrong and
wasting time and expensive medium.
Any suggestion help, so if you know what I am doing wrong, or how it suppose to looks like, let me know!!!
Thanks in advance
Posted 26 January 2011 - 12:11 PM
It seems that you are doing everything the way it is supposed to be.
The intermediate colonies usually look just like...
ta da, regular ES colonies but a little more disperse.
We tend to use Nagog or Oct4 reporter cell lines to make it easier to determine if the cell reached pluripotency or not.
The hardest thing in IPS generation is all the virus work and the myc titers. Once you get these right I think your set.
(post some pictures of your cells)
Posted 16 December 2012 - 10:00 PM
typically you should see isolated colonies or clusters of cells on the MEF feeder in 7-10 days. Here are typical images of mouse iPS cells