Human iPSC culture

All the experiments involving hiPSCs were approved by the ethics committee of Kansai Medical University (Approval Number: 2020197). We obtained the written informed consent of the donors from whom hiPSCs were derived. The study was performed according to the principles of the Declaration of Helsinki, as revised in 2013, and relevant institutional guidelines. Human iPSCs (585A1, 253G1, and HiPS-RIKEN-2F) were maintained with feeder-free cells using NutriStem hPSC XF (05-100-1A, Sartorius AG, Goettingen, Germany) on plates coated with iMatrix-511 silk (892021, Matrixome, Osaka, Japan) at 37C in a 5% CO2 incubator. Single cells were prepared from hiPSC colonies (7090% confluent) using Accutase (AT104, Innovative cell technologies, CA, USA) for subsequent passage and the induction of podocyte differentiation.

We generated podocytes from hiPSCs by modifying a previously reported differentiation protocol16 (Fig.1A). Human iPSCs were seeded at 3000 cells/well in 96 well low-cell-binding V-bottom plates, which were cultured in 200L NutriStem medium containing 10M Y27632 (FCS-10-2301-25, Focus biomolecules, PA, USA) at 37C for 24h. The medium was changed to DMEM Hams/F12 medium (048-29775, Fujifilm, Osaka, Japan) containing 2% B27 supplement (17504044, Thermo Fisher Scientific, MA, USA), 1ng/mL human activin A (338-AC, R&D Systems, MN, USA), and 20ng/mL fibroblast growth factor 2 (FGF2, 064-04541, Fujifilm). After 24h, cell aggregates were cultured for 6days in a medium (DMEM Hams/F12 medium) containing 2% B27 supplement and 10M CHIR99021 (10-1279, Focus biomolecules) that was changed every 2days. Subsequently, the medium was changed to one containing 10ng/mL human activin A, 3ng/mL human bone morphogenetic protein 4 (BMP4, PROTP12644, R&D System), 3M CHIR99021, and 100nM retinoic acid (RA, 302-79-4, Fujifilm). After a further 72h, this medium was switched to one containing 1M CHIR99021 and 10ng/mL FGF9 (273-F9, R&D Systems) without medium change to induce the differentiation of NPCs.

Differentiation of hiPSCs into podocyte. (A) Timeline and factors involved in the differentiation of hiPSCs into podocytes. (B) mRNA expression of podocyte-associated genes (NEPHRIN, PODOCIN, and SYNAPTOPODIN) during the 24days of culture. Results are shown as the meanSD of 6 samples. Statistical analysis was performed using one-way ANOVA with Bonferronis test. **p<0.01, ***p<0.001. (C) Immunostaining for markers of podocytes (NEPHRIN and PODOCIN) and F-Actin in differentiated cells, with nuclei stained with Hoechst. (D) mRNA expression of podocyte-associated genes (NEPHRIN, PODOCIN, and SYNAPTOPODIN) in hiPSCs, NPCs and differentiated podocytes. Results are shown as the meanSD of 6 samples. Statistical analysis was performed using one-way ANOVA with Bonferronis test. *p<0.05 (E) Protein expression of nephrin and podocin in hiPSCs, NPCs and differentiated podocytes, assessed using western blotting analysis. (F) Protein expression of undifferentiation stem cell marker (OCT-3/4) and nephron progenitor cell marker (SIX2) in hiPSCs, NPCs and differentiated podocytes, assessed using western blotting analysis. (G) Protein expression of nephron progenitor cell marker (SIX2) assessed using western blot analysis. Results are shown as the meanSD of 3 samples. Statistical significance was assessed using Students t-test. *p<0.05.

To generate podocytes, the medium was switched to one containing 3M CHIR99021, and after 24h, to one containing 2M IWR-1 (1127442-82-3, Fujifilm), 5M SB431542 (13031, Cayman Chemical, MI, USA), and 10M RA. After a further 24h, the differentiated cells were cultured for 11days in fresh medium containing 2M IWR-1 and 5M SB431542, which was replaced every 3days. Cell sorting was not performed at all steps.

To construct the monolayer cell culture, the cell aggregates were transferred to a 50-mL centrifuge tube, washed with PBS, then dissociated using Accutase. The cells (2,000 cells/cm2) were then seeded onto iMatrix-511 silk-coated dishes and cultured in DMEM Hams/F12 medium supplemented with 10M Y27632 and 2% B27 supplement. Cells were collected 24h after the treatment with DMEM Hams/F12 medium supplemented with Y27632 and B27 supplement.

To evaluate the involvement of the mTOR pathway in podocyte differentiation, rapamycin (R0161, LKT Laboratories, MN, USA) was administered at various times during the differentiation process and evaluated by mRNA expression using RT-PCR. In addition, S6 downstream of mTOR was inhibited using LY2584702 to further assess its involvement in the mTOR pathway.

RNA was extracted from the cells using ISOGEN II reagent (311-07361, Nippon gene, Tokyo, Japan), then a ReverTra Ace qPCR RT Master Mix (FSQ-201, Toyobo, Osaka, Japan) was used for reverse transcription. Real-time PCR was performed to quantify target mRNA expression using a Rotor-Gene Q (Qiagen) and Thunderbird SYBR qPCR Mix (QPS-201, Toyobo). The specific PCR primers used are listed (Table 1).

Cell lysates were collected using 4Bolt LDS Sample Buffer (B0007, Thermo Fisher Scientific), then electrophoresed on a 10% SDS polyacrylamide gel and blotted onto PVDF membranes. The membranes were incubated with anti-NEPHRIN (29070, Immuno-Biological Laboratories, Gunma, Japan), anti-PODOCIN (MBS9608910, Thermo Fisher Scientific), anti-Phospho-Akt (9271, Cell Signaling Technology, MA, USA), anti-Akt (9272, Cell Signaling Technology), anti-Phospho-mTOR (2971, Cell Signaling Technology), anti-mTOR (2972, Cell Signaling Technology), anti-Phospho-p70 S6 Kinase (9205, Cell Signaling Technology), anti-p70 S6 Kinase (2708, Cell Signaling Technology), anti-Phospho-S6 Ribosomal Protein (2211, Cell Signaling Technology), S6 Ribosomal Protein (2217, Cell Signaling Technology), anti-SIX2 (80170, Cell Signaling Technology), anti-OCT3/4 (611202, BD Biosciences, NJ, USA), and anti- actin (MAB8929, R&D Systems) primary antibodies, then further probed with anti-mouse IgG horseradish peroxidase-linked (A90-131P, Bethyl Laboratories, TX, US) secondary antibody. Specific protein bands were visualized using Pierce Western Blotting Substrate (NCI3106, Thermo Fisher Scientific).

Cultured cells were harvested after detachment using Accutase, then incubated for 30min at 4C with FITC-conjugated anti-PODOCIN antibody diluted 1:20. The cells were then centrifuged, the supernatants removed, and 500-L aliquots of PBS containing 2% StemSure Serum Replacement (191-18375, Fujifilm) added. Data were acquired using a BD FACS Canto II flow cytometer system (BD Biosciences).

Cells were fixed using 4% paraformaldehyde, and blocked with Blocking One (03953-95, Nacalai Tesque, Kyoto, Japan) for 60min at room temperature. Incubations were then performed at 4C overnight using primary anti-NEPHRIN, anti-PODOCIN antibody, and F-Actin (bs-1571R, Bioss Inc., MA, USA) antibody. Then, Alexa Fluor 488-tagged secondary antibody (ab150107, Abcam, Cambridge, UK) was applied for 30min at room temperature, and nuclei and F-actin were stained using 10g/mL Hoechst 33342 (346-07951, DOJINDO Laboratories, Kumamoto, Japan) and Phalloidin-iFluor 647 Conjugate (23127, AAT Bioquest, CA, USA), respectively. The stained cells were evaluated using fluorescence microscopy (BZ-X810, Keyence, Osaka, Japan).

Podocytes differentiated from hiPSCs were seeded at 2000 cells/cm2 onto Transwell inserts in six-well culture plates, pore size 0.4m (3450, Corning, AZ, USA) coated with iMatrix-511 silk. After 24h, DMEM Hams/F12 medium containing 2% B27 supplement, potassium chloride (5mM), urea (25mg/L), and human serum albumin (3g/dL) were added to the lower chambers, whereas the cells were incubated in a medium lacking the latter three substances in the upper chambers. After 24h, the media were collected from both of the chambers. The potassium concentration was measured using reagent for potassium measurement and electrode (EA09, A&T Corporation, Kanagawa, Japan). The urea nitrogen and albumin were measured using CicaLiquid-N UN reagent (77697, Kanto Chemical, Tokyo, Japan) and reagent of modified BCP method for albumin (30155001, Sekisui Medical, Tokyo, Japan), respectively, by an autoanalyzer (JCA-BM8020, JEOL Ltd., Tokyo, Japan).

Data are expressed as meanstandard deviation (SD). All experiments resulted by repeating the experiment three independent times. For the results shown in Figs.1B, 2A, and 3B, statistical analysis was performed using one-way ANOVA, followed by Bonferronis test; and Students t-tests were performed to compare the mean values of two groups for the data shown in Figs.2C and 5B. A p-value of<0.05 was considered to indicate statistical significance.

Effects of an mTOR inhibitor on podocyte differentiation. (A) Evaluation of the timing of rapamycin administration for protocol improvement: (a)13days treatment, (b)11days treatment and (c)7days treatment. (B) mRNA expression of podocyte-associated genes (NEPHRIN, PODOCIN, WT1, and MAFB) in cells treated with 100nM rapamycin at different times (a, b, c). Results are presented as meanSD of 6 samples. Statistical analysis was performed using one-way ANOVA with Bonferronis test. *p<0.05, **p<0.01. (C) mRNA expression of podocyte-associated genes (NEPHRIN, PODOCIN, SYNAPTOPODIN, WT1, and MAFB) in cells treated with various concentrations of rapamycin. Results are shown as the meanSD of 6 samples. Statistical analysis was performed using one-way ANOVA with Bonferronis test. *p<0.05, **p<0.01, ***p<0.001. (D) Protein expression of nephrin and podocin in differentiated podocytes, assessed using western blotting analysis. (E) Protein expression of nephrin and podocin assessed using western blot analysis. Results are shown as the meanSD of 3 samples. Statistical significance was assessed using Students t-test. *p<0.05. (F) Histograms for podocin-positive cells, quantified using FACS: (a) undifferentiated hiPSCs and (b) podocytes differentiated from hiPSCs.

Importance of the mTOR pathway for podocyte differentiation. (A) Protein expression of mTOR, p-mTOR, p70 S6K, p-p70 S6K, S6, p-S6, AKT, and p-AKT, assessed using western blotting analysis. (B) mRNA expression of podocyte-associated genes (NEPHRIN, PODOCIN, SYNAPTOPODIN, WT1, and MAFB) following the addition of the S6 inhibitor LY2584702. Results are shown as the meanSD of 6 samples. Statistical analysis was performed using one-way ANOVA with Bonferronis test. ***p<0.001.

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