I have created over a thousand human iPSC clones but only monitored the reprogramming of a few murine iPSC lines, so my experience is limited.

Survival will be better on feeder cells, but feeders do not survive well in KOSR medium.  I don't know if is still readily available, but Plasmanate replacement of half of the KO serum drastically increases survival of feeders in KOSR medium.  In fact, you can freshly plate feeders in Plasmanate supplemented medium, while they will fail to attach in straight KOSR medium.

You also may want to consider replacing the gelatin with matrigel for better survival. 

Another issue for reprogramming success is the density of the cells during the reprogramming phase.  I have not experimented with this with murine reprogramming but for human reprogramming the requisite density of the cells is highly variable depending on the reprogramming media used.  For example, E7 requires a very high density while Pluriton requires more than an order of magnitude fewer cells for optimal reprogramming recovery.  For this reason, I would speculate that you may benefit by attempting a reprogramming with a variety of densities to find the one that works best for your conditions.

Dear Ron and Antonio,

Thank you so much for your reply. Good to know it's not my problem that my feeder cells do not survive well on KOSR. I'm not quite sure if I can get that plasmanate easily, but do you think I can slowly adapt my cells to KOSR by changing half of the medium everyday with KOSR?

I will have to make human iPSC in future after I proof my hypothesis using the mouse system, can I ask for your advice as well? My goal is to reprogramme cancer cells and my problem (also with mouse cells) is overtaking by parental untransfected cells. I have only made several attempts to reprogramme my cells, so it can be my lack of experience. I thought that plating transfected cells on feeders at too low a density would be detrimental, but when I plated them at high densities, the cells became confluent in just 2-3 days and I would have to split them even before the emergence of any iPSC-colonies. Should I try to plate less cells after transfection? Or should I leave them be (but the medium would turn yellow even with daily changes)?

Antonio, thanks for your information. What is the concentration of KOSR you are using?

Sorry, but I have to start with two questions before addressing the issue more.

1.  Are you committed to the KOSR-based reprogramming media?

2.  Are you required to prove successful murine reprogramming before moving on to human?  Given that they behave differently, optimizing one will not necessarily benefit doing the other.

Dear Antonio and Ron,

Thanks for the reply.

For now I would have to stick with protocols with the use of feeders. I know that mTeSR is a more commonly used medium base now, and so is E8 medium. I am using KOSR-medium because my predecessor was using it. But if other medium system is better I can change. 

I do understand that reprogramming human and mouse cells should require very different protocols, but I would need to use a mouse disease model first for testing, so even though it's so very true optimizing mouse protocol will not necessarily help in optimizing the human protocol, I will still have to do it.