Isolation and purification of urine cells

The study protocol for human urine sample collection was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University, and Guangzhou Institutes of Biomedicine. Informed consent was obtained from all participants. Urine Cells were collected following the protocol described [13]. The samples of the mid-stream of urine from healthy female and male volunteers were collected and stored at 4 °C within 8 h before purification. The usual volume of specimens was 50–200 mL. The samples were centrifuged at 500 g for 8 min, and then the pellet was washed with about 10 mL DPBS supplemented with 1 × penicillin/streptomycin (lot. SH40003.01, Hyclone). After another centrifugation, the supernatant was removed and the pellets were resuspended with the urine cell culture medium supplemented with 10 μg/mL Primocin (lot. ant-pm-1, Invivogen). The urine cell culture medium was a one-to-one mixture of Renal Epithelial Growth Medium (REGM; lot. CC-3191, Lonza) and DMEM High Glucose (lot. SH30022.01, Hyclone) containing 10%FBS, 1 × L-GlutaMAX (lot. 35050-061, GIBCO), and 1 × non-essential amino acids (NEAA; lot. 11140-050, GIBCO). Then the pellets were plated in the gelatin (lot. 07903, STEMCELL) coated 6 cm culture dish and the culture medium was changed after 4 days. A few days later, hUCs grew and formed a few clusters of uniform colonies. In about 1–2 weeks, hUCs achieved confluence thereby allowing us to passage and perform reprogramming experiments.