Hello Soo hyun.

Hard to tell without looking at the extent of the reality.

Preparation of mitomycin C-treated cells should be firm attachment to the bottom of the dish, confluent (80~100%), and free of mitomycin C after several hours of the treatment by lot of gentle PBS washings to eliminate toxcity carried over to iPSCs. Better to use 5~6 days alone to get maximum release of growth factors for the feeder layer activity from the mitomycin C-treated cells. Induced differentiation causes many cell death due to the arrested cell cycle for cell differentiation.

The OP9 cells should feed iPSCs with enough number and fresh activity with no detached or lysing or lysed cells.

Best wishes.

Hello Sang Ho,

Thank you for your answer. After 2.5 to 3 hr mitomycin C treatment, I usually wash cells with PBS three times, treat them with Trypsin and replate them on 0.1% gelatin coated plate. Next day, I plate iPSCs on top of OP9 cells. Because I detected massive cell death, I replated differentiating iPSCs on new OP9 feeder cells every 3-4 days for 10 day differentiation. In this way, I could get only a few of differentiated cells from iPSCs. However, I couldn't see colonies derived from iPSCs because cells were dissociated by trypsin. So, I was wondering how I could co-culutre iPSC with OP9 cells without replating them and see the colony formation, which may increase the number of CD34+ cells at the end. I will try your suggestions. Let me know if you have any other suggestions. Thank you so much.

Hello Soo hyun.

Try to reduce changes of the stem cell niche formed upon differentiation signals or any further stresses that may be pressed on either feeder layer (already feeble cell layer) or iPSCs.

I noticed from what you described above that you could avoid what I called changes and stresses in the culture system.

1) Avoid T-E digestion on OP9 and possible stresses from trace amount of mitomycin C to either feeder layer or iPSCs.

After full confluency of OP9 cells on already attached on gelatin-coated-dish, treat mitomycin C in the same dish. Wash with PBS 3x and then replace with enough medium, back to incubator for 2~3h to release any remaining trace of mitomycin C. Wash with medium 3x.

2) Avoid 3 changes, you could get away with the above-prepared feeder layer not to disturb the stem cell niche formed during last 3 days, extending to 5~6 days.

Seed enough number of iPCSs (excess would be a good asset) directly on the feeder layer dish for 5~6 days to grow any colonies formed. Thus, you are changing feeder just once after 5~6 days, replating differentiating iPSCs from 3~4 dishes into one new feeder dish (3~4 vs 1 dish) to maintain appropriate cell numbers for their cross-talks at over the threshold concentrations of goodies released from the cells themselves.

Good luck.

Hello Sang Ho,

Thank you so much for detailed explanation. I will definitely try your suggestions. Thank you again.

Soo