In my opinion, you don't have any iPSCs here yet. iPSCs need to form round-shaped colonies which grow as a ball, not as flat thing. They need to have clearly defined borders, and a characteristic color that is sometimes called "glow".

However, you absolutely need to confirm the success of reprogramming by at least AP (alkaline phosphatase) staining - it is very easy, just get a kit, it can be done in 30 mins. It will also help you to understand which stage of reprogramming you are at. I attach two pics, one is just microscopy that shows what a good colony looks like, and the second is AP staining showing how the color of the stain appear gradually as the reprogramming goes on.

Thank you Yulia Panina ! It's really helpful. I have 2 more relative questions:

1) In your picture, density at Day 1 and 10 is not much higher. My cells are continuously growing and it is going to 100% confluency with the passage of time. Is it ok for the reprogramming process to have a fully confluent well?

2) Do you add new feeder cells to the well after every 1 week to keep the population normal as some of the feeder cells might die and the overall percentage of growth factors will be declining?

I am happy to help!

1) The speed of cell multiplication really depends on your cell type. Some grow faster than others. In my case, these pictures are of neural progenitor cells. Your cells may multiply faster. Now, it has been shown in several papers that the success of reprogramming depends on cell confluency. If cells become too confluent, you will not be able to reprogram them. To identify ideal cell density, it would be useful to plate several plates with different cell quantities, and then see which ones form colonies. 100% confluency should be avoided! Or you will not be able to reprogram. Plate less cells in the beginning.

2) In my experience, feeder cells may not be necessary. I would try to reprogram your cells without feeders first. You can use "Essential 8" medium which is designed for reprogramming and has growth factors needed for it. At the start of reprogramming, change the medium to Essential 8 and then use it for the whole process. (Although, it may be that your cell type will not tolerate it; then you would need another solution).

I also noticed that it is absolutely necessary to monitor PH. Normally, when reprogramming starts, PH will start to change, so your normally pink medium will become yellow-ish. This has to be avoided. In my case, I change the medium every day for smooth and nice reprogramming, and never let the medium become fully yellow.

I am also changing the medium regularly on daily basis but still it becomes yellow (might be because of the higher density). I will optimize the density and follow your recommendations. Thank you again Yulia Panina !

Dear Nasir,

I am interested in reprogramming HaCaT cells. Would you mind if you share your protocol with me? I've tried the conventional method of transducing Sox2, Oct4 and Klf4 but cells were too stressed and detached by week 1.

Thank you