Cell culture

Epithelial cells were isolated from urine samples according to the protocol described previously [10]. Briefly, 100 mL of urine sample was collected, transferred into a 50-mL conical tube and centrifuged at 400×g for 10 min at room temperature. The supernatant was removed; the cell pellet was washed twice with 25 mL of PBS supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin B (0.25 µg/mL) and centrifuged again. The supernatant was discarded, and the cell pellet was suspended in Renal Epithelial Cell Growth Medium (REGM BulletKit, Lonza) and plated on gelatine-coated cell culture plates (Attachment Factor Protein, Life Technologies). After reaching 90% confluency, cells were passaged with TrypLE Select (Life Technologies) into a new well for further expansion.

Induced pluripotent stem cells were cultured according to Drozd et al. [9].

Reactive oxygen species detection

A cellular reactive oxygen species (ROS) assay kit (Abcam, ab186027) was used to determine ROS levels, according to manufacturer’s protocol. Statistical analysis was performed using GraphPrism 5 software. Comparisons among groups were performed using One-way ANOVA with Dunnett’s multiple comparison test. Error bars indicate SD (n = 3). P < 0.05 was considered statistically significant.

Reprogramming of urinary epithelial cells into iPSc and following differentiation

IPS cells were generated as described previously [9]. Urinary epithelial cells were seeded at a density of 8 × 104 per well of a six-well plate coated with Geltrex basement matrix. Cells were maintained in REGM medium. After overnight incubation, the culture medium was replaced with a fresh one, and cells were transfected with 2 μg of episomal plasmids (Epi5™ Episomal iPSC Reprogramming Kit, Life Technologies), 400 ng of each: pCE-hOct3/4, pCE-hSK pCE-hUL, pCE-mp53DD, pCXB-EBNA1 and 1 μg of an additional plasmid to increase efficiency—pCE-mCherry-miR302/367. FuGENE HD transfection reagent (Promega), at a 3:1 reagent-to-DNA ratio, was diluted in pre-warmed Opti-MEM medium and incubated for 5 min at room temperature. Plasmid DNA was added to the mixture up to a total volume of 100 μL and incubated for 30 min. The solution of the complexes was added in a dropwise manner directly to cells grown in one well of a six-well plate in 2 mL of medium.

The next day, the culture medium was replaced with TeSR-E7 medium (StemCell Technologies), and the transfection was repeated as previously. TeSR-E7 medium was changed every day up to two weeks post-transfection. On day 15, the culture medium was changed to Essential 8. The medium was replaced daily for the next two weeks. Within twenty to thirty days post-transfection, iPSCs expanded to a size appropriate for transfer. Colonies were transferred onto new Geltrex-coated culture dishes and further propagated in Essential 8 medium.

Finally, differentiation of iPS cells into three germ layers was conducted as described previously [9].

Immunofluorescence analysis

For the immunocytochemical analyses, iPSc or other cells were seeded on Geltrex-coated glass coverslips. Cells were fixed in 4% paraformaldehyde in PBS for 20 min (iPSc) or 15 min (differentiated cells) and permeabilized with 0.25% (iPSc) or 0.1% (differentiated cells) Triton X-100 in PBS for 10 min at room temperature. Next, preparation was performed as already described [9] (Table 1).

Table 1 Antibodies used for immunocytochemical staining

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