iPS induction difficulty from MEF - how they suppose to look like???? (Nov/06/2010 )
Hi, I have a little difficulty to making iPS from MEF culture, so I would like to ask question to the experts here.
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
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)
Can anybody tell why the authors used mouse transcription factors (OCT4, SOX2, KLF, LIN28 & c-MYC) for reporgramming human cells in the following paper? http://www.nature.com/cr/journal/v21/n3/full/cr201112a.html
EBNA1/OriP episomal vectors
Mouse iPS induction should be easier than human. Since you are using the standard platform which should work as expected. However the critical part is the quality of your virus and the transduction efficiency. Do you have a sense of your transduction efficiency and the expression of exogenous genes upon induction by doxycycline?
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