Optimized Approaches for Generation of Integration-free iPSCs from Human Urine-derived Cells with Small Molecules and Autologous Feeder

We generated iPSCs from human urine cells (hUCs) with the aid of small molecules and autologous hUC feeders. A compound cocktail including Cyclic Pifithrin-a, a p53 inhibitor and other compounds known for benefiting reprogramming like A-83-01, CHIR99021, Thiazovivin, NaB and PD0325901 was used to aid hUC reprogramming (Plan B). Aided by this cocktail, we achieved significantly improved efficiency (170 folds more) for hUC reprogramming and iPSC generation. In addition, to enable iPSC generation in some cases that massive cell death occurred during delivering reprogramming factors, we replaced Matrigel with autologous hUCs as feeder for reprogramming and iPSC generation (Plan C). Replacing Matrigel with autologous feeder not only enhanced reprograming, but also avoided concern using animal components for human iPSC generation. These were efficient approaches to enable iPSC generation from hUCs that were otherwise difficult for reprogramming, which would be valuable for banking patient’s specific iPSCs. Overall design: We compared the transcriptome of hESCs, hiPSC, hUC by RNA-Seq. hiPSCs were generated from Plan B and Plan C. Plan B: RM at day 0~1, RM+5M at day 2~9, mTeSR1+6M at day10~17. UCs were seeded on Matrigel. Plan C: Medium was the same with Plan B, but seeded on autologous UCs as feeders before nucleofection.