Generation of Patient-Derived Podocytes from Skin Biopsies

The protocol was approved by the ethics committee of Friedrich-Alexander University Erlangen-Nuremberg (251_18B), and all patients and probands gave written consent. All experiments were performed in accordance with relevant guidelines and regulations. For composition of all media and solutions used here see Table 1.

1. Outgrowth of dermal fibroblasts from a skin punch biopsy

1. Transfer the skin punch biopsy to a sterile 15 mL conical tube with 10 mL of prewarmed fibroblast medium.
2. Remove the medium and wash the skin punch biopsy three times with 5 mL of sterile, prewarmed 1x phosphate-buffered saline (PBS). Transfer the skin punch biopsy with sterile forceps to a 10 cm cell culture plate and cut it widthwise in three or four pieces with a sterile scalpel, leaving the epidermis and dermis.
3. Transfer each piece to a sterile 35 mm plastic cell culture dish and press it gently to the culture dish. Remove excess medium around the biopsy pieces. Allow to dry for 5-10 min until the liquid evaporates and the biopsy is attached to the cell culture plastic.
4. Add 1 mL of fibroblast medium dropwise with a 1,000 µL pipette around the biopsy and fill up carefully to a final volume of 3 mL. Cultivate at 37 °C, 5% CO₂ for 7 days without feeding and moving the dish. Carefully change the medium to fresh, prewarmed fibroblast medium. After 7 days, outgrown spindle-like fibroblasts can be monitored around the skin biopsy using a phase contrast microscope³⁶.
   NOTE: When outgrown fibroblasts reach the rim of the dish, proceed again from step 1.3 to generate another batch of fibroblasts.
5. For expansion of the outgrown fibroblasts, wash with 2 mL of prewarmed 1x PBS, dissociate the fibroblasts with 1 mL of 1x trypsin-ethylenediaminetetraacetic acid (EDTA) and incubate for 5 min at 37 °C, 5% CO₂. Transfer the detached cells to a 15 mL conical tube and wash the cell culture dish with 2 mL of fresh, prewarmed fibroblast medium.
6. Pool the washing medium in the tube to neutralize the dissociation agent and centrifuge at 200 x g for 5 min at 20 °C. Aspirate the supernatant and resuspend the cell pellet with 1 mL of fresh, prewarmed fibroblast medium. Count the cells with a Neubauer chamber or automated cell counter using trypan blue and seed 2.5 x 10³ to 5 x 10³ fibroblasts per cm² in fresh cell culture flasks.
7. Place the flasks back in the incubator and distribute the cells evenly by moving the flasks three times in each direction. The next day, replace the medium with fresh, prewarmed fibroblast medium. Change the medium twice a week and split when the fibroblasts reach around 80% confluency.

2. Freezing dermal fibroblasts

1. To freeze the fibroblasts, wash them with 5 mL of prewarmed 1x PBS. Aspirate the PBS and dissociate the fibroblasts with 4 mL of 1x trypsin-EDTA. Place the flask back in the incubator and incubate for 5-7 min at 37 °C at 5% CO₂. Monitor detachment using a phase contrast microscope and tap against the side of the flask to detach the cells.
2. Transfer the detached cells to a 15 mL conical tube. Wash the cell culture flask with 5 mL of fresh, prewarmed fibroblast medium. Pool the washing medium in the conical tube and centrifuge at 200 x g for 5 min at 20 °C.
3. Aspirate the supernatant and resuspend the cell pellet with 2 mL of fresh, prewarmed fibroblast medium. Count the cells and transfer 1 x 10⁶ cells to a new conical tube. Centrifuge at 200 x g for 5 min at 20 °C.
4. Aspirate the supernatant and resuspend the cell pellet with 1 mL of cold fibroblast freezing medium consisting of 90% fetal calf serum and 10% dimethyl sulfoxide (DMSO). Transfer to a cryovial, place in a freezing container, and freeze overnight at -80 °C. For long term storage, place the cryovial in a liquid nitrogen tank the next day.

3. Episomal reprogramming of fibroblasts to generate hiPSCs

1. For episomal reprogramming, use 1.5 x 10⁶ fibroblasts from passage 4 to 8. To reach this cell number, use two to three 250 mL flasks with around 70% confluency.
2. The day before reprogramming, split the fibroblasts in a 1:2 ratio to ensure their proliferative state. Therefore, aspirate the medium and wash the flasks with 5 mL of prewarmed 1x PBS per flask. Aspirate the PBS and dissociate the fibroblasts with 4 mL of 1x trypsin-EDTA. Place the flasks back in the incubator and incubate for 5-7 min at 37 °C and 5% CO₂. Monitor detachment using a phase contrast microscope and tap against the side of the flask to detach the cells from the plastic surface.
3. Transfer the detached cells to a 50 mL conical tube and wash the cell culture flasks with 5 mL of fresh, prewarmed fibroblast medium. Pool the washing medium in the conical tube and centrifuge at 200 x g for 5 min at 20 °C. Aspirate the supernatant and resuspend the cell pellet in 6 mL of fresh, prewarmed fibroblast medium.
4. Prepare six new cell culture flasks by adding 5 mL of fresh, prewarmed fibroblast medium per flask. Add 1 mL of fibroblast cell suspension to each flask. Place the flasks back in the incubator and distribute the cells evenly by moving the flasks three times in each direction.
5. For episomal reprogramming, coat two 10 cm cell culture plates suitable for hiPSC culture with 4 mL of cold coating solution per plate. Incubate the plates for 1 h at 37 °C.
   NOTE: To culture hiPSCs, different cell culture plastics and additional extracellular matrix coating are required (see Table of Materials).
6. Remove the media from the fibroblasts and wash with 10 mL of prewarmed 1x PBS per flask. Aspirate the PBS and dissociate the fibroblasts with 4 mL of 1x trypsin-EDTA per flask by incubating for 5-7 min at 37 °C. Monitor detachment using a phase contrast microscope and, if necessary, tap against the side of the flask to detach the cells from the plastic surface.
7. Transfer the detached cells to a 50 mL conical tube. Wash the empty flasks with 6 mL of prewarmed fibroblast medium to collect the remaining cells and pool in the conical tube. Centrifuge at 200 x g for 5 min at 20 °C. Aspirate the supernatant and resuspend the cell pellet in 3 mL of fresh, prewarmed 1x PBS.
8. Count the cells and transfer 1.5 x 10⁶ fibroblasts in a new conical tube. Centrifuge at 200 x g for 5 min at 20 °C. Discard the supernatant and resuspend the cell pellet in 5 mL of electroporation medium and centrifuge again. In the meantime, aspirate the coating solution from the coated 10 cm cell culture plates and add 7 mL of prewarmed fibroblast medium.
9. After centrifugation, discard the supernatant and resuspend the cell pellet in electroporation medium with a concentration of 1.5 x 10⁶ cells in 250 µL. Transfer the 250 µL cell suspension to an electroporation cuvette with a 4 mm gap distance (see Table of Materials).
10. Prepare a plasmid transfection mix by adding 4 µg of each plasmid (pCXLE-hOCT3/4, pCXLE-hSK, pCXLE-hMLN) to a total volume of 50 µL of electroporation medium. Transfer to the cuvette and mix by flicking gently. Electroporate with one pulse at 280 V.
11. Cut a pipette tip using sterile scissors and transfer 125 µL of the electroporated fibroblasts to each of the prepared 10 cm cell culture plates. Distribute the cells by agitating the plates three times in all directions and place them back in the incubator. Incubate overnight at 37 °C with 5% CO₂ without disturbance.
12. To remove dead cells, replace the medium the next day with 7 mL of fresh, prewarmed fibroblast medium. Change the medium to hiPSC culture medium 2 days after electroporation and replace it every other day for the next 20 days.

4. Selection, expansion, and quality control of generated hiPSCs

1. Monitor the cells daily for at least 20 days using a phase contrast microscope with a 10x or 20x objective to observe the formation of hiPSC colonies after electroporation. If hiPSC colonies are around 300 µm diameter in size with distinct borders and the hiPSCs show a high nuclei-to-body ratio, the hiPSC colonies are ready for selection by picking (Figure 2B,C and Figure 3).
2. Prior to picking, coat a 96-well plate suitable for hiPSC culture with 100 µL of coating solution per well and incubate for 1 h at 37 °C. During incubation, mark colonies of interest at the bottom of the cell culture dish with a pen. For final preparation of the 96-well plate, remove the coating solution and add 100 µL of prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632 to each well.
3. Wash the cells with prewarmed 1x PBS and add fresh, prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632 before picking to remove dead cells. To pick hiPSC colonies, use a gauge needle and divide the hiPSC colonies into small pieces by drawing a grid into each colony.
4. Using a phase contrast microscope, check that the colonies are successfully divided into pieces. Transfer them with a 100 µL pipette to the prepared 96-well plate. Hold the pipette upright over the colony without touching the cells to avoid scratching and loss of the colony.
5. Place the 96-well plate in the incubator at 37 °C with 5% CO₂ and let the cells attach overnight without disturbance. Keep the dishes with the fibroblast and hiPSC mix, in case picking is not successful. Therefore, remove ROCK inhibitor Y27632 by changing the medium to hiPSC culture medium and place the plates back in the incubator.
6. The next day, change medium of the 96-well plate to 200 µL of fresh hiPSC culture medium and change it every other day. Monitor the 96-well plate the next day and mark the wells with successfully picked clones.
   ​NOTE: If the picked hiPSC clones are not fully selected after the first try and the fibroblasts can be found in the well, repeat picking from the 96-well or scrape the fibroblasts off the plate.

5. Expansion of selected hiPSC clones

1. Monitor the hiPSC clones using a phase contrast microscope. If the selected hiPSCs reach around 70% confluency, the hiPSC clones are ready for expansion.
2. Coat a 48-well plate suitable for hiPSC culture with 250 µL of coating solution per well for 1 h at 37 °C. Replace the coating solution with 200 µL of fresh, prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632.
3. To detach the hiPSCs from the 96-well plate, scrape the plastic surface with a 1,000 µL pipette tip. Transfer the detached hiPSCs from the 96-well plate to a 48-well plate. Change the medium the next day to fresh, prewarmed hiPSC culture medium to remove dead cells and the ROCK inhibitor Y27632.
4. If the hiPSC clones reach around 70% confluency, transfer the hiPSCs into a 24-well plate. Therefore, coat a 24-well plate suitable for hiPSC culture with 400 µL of coating solution per well for 1 h at 37 °C. Replace the coating solution with 400 µL of fresh, prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632. Aspirate the medium from the 48 wells and wash with 500 µL of prewarmed 1x PBS.
5. Replace the PBS with 100 µL of enzymatic cell detachment solution containing 10 µM ROCK inhibitor Y27632 and incubate at 37 °C for 4 min. Rinse the hiPSCs off the plate using a 1,000 µL pipette. Transfer the dissociated cells to a 15 mL conical tube. Wash the empty 48-well plate with hiPSC culture medium containing 10 µM ROCK inhibitor Y27632 and pool in the conical tube.
6. Centrifuge at 200 x g for 5 min at 20 °C. Aspirate the supernatant and resuspend the hiPSCs in 1 mL of hiPSC culture medium containing 10 µM ROCK inhibitor Y27632 and transfer to a 24-well plate. Place the plate in the incubator at 37 °C at 5% CO₂ and distribute the cells by agitating the plate three times in all directions. Let the cells attach overnight without disturbance.
7. The next day, change the medium to hiPSC culture medium without ROCK inhibitor Y27632.
   ​NOTE: If hiPSC clones reach around 70% confluency, transfer the hiPSCs to a 12-well plate by repeating steps 5.4 to 5.7 with increased volumes suitable for a 12-well format. If the hiPSCs from the 12-well plate reach around 70% confluency, transfer the hiPSC clones to a 6-well plate with increased volumes for a 6-well format.

6. Freezing of selected hiPSC clones

1. To freeze selected hiPSC clones, aspirate the medium from a 6-well plate. Wash the wells with 2 mL of 1x PBS. Aspirate and dissociate the hiPSCs with 1 mL of enzymatic cell detachment solution containing 10 µM Y27632. Place the plate back in the incubator and incubate for 4 min at 37 °C and 5% CO₂.
2. Rinse the hiPSCs off the plate using a 1,000 µL pipette and transfer the detached cells to a 15 mL conical tube. Wash the plate with 2 mL of fresh, prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632. Pool in the conical tube and centrifuge at 200 x g for 5 min at 20 °C. Aspirate the supernatant and resuspend the cell pellet with 2 mL of fresh, prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632.
3. Count the cells and transfer 1 x 10⁶ cells to a new conical tube. Centrifuge at 200 x g for 5 min at 20 °C. Aspirate the supernatant, resuspend the cell pellet in 1 mL of cold, serum-free hiPSC freezing medium, and freeze in a cryovial using a freezing container overnight at -80 °C. For long-term storage, place the cryovial in a liquid nitrogen tank the next day.

7. HiPSC quality control

1. Characterization of hiPSC morphology
   1. Check the characteristic hiPSC morphology using a phase contrast microscope with a 20x objective. After reprogramming, the cell morphology changes from long, spindle-like fibroblasts to small, round hiPSCs, presenting a high nuclei-to-body ratio growing in colonies with distinct borders (Figure 2C).
   2. If the hiPSC colonies grow too dense and spontaneous differentiation occurs, scrape the differentiated parts off the plate using a pipette tip (Figure 3). Since hiPSCs have a high proliferation rate, feed the hiPSC cultures every day with fresh, prewarmed hiPSC feeding medium.
2. Characterization of pluripotency markers by immunofluorescence staining
   1. To check the generated hiPSCs for pluripotency marker via immunofluorescence staining, place plastic coverslips in a 24-well plate and coat with 250 µL of coating solution for 1 h at 37 °C and 5% CO₂. Replace the coating solution with 1 mL of prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632.
   2. Seed 1.9 x 10⁴ dissociated hiPSCs per 24-well. Let the hiPSCs attach overnight at 37 °C and 5% CO₂ without disturbance. See steps 5.4 to 5.6 for dissociation of the hiPSCs. Replace the medium the next day with hiPSC culture medium without ROCK inhibitor Y27632.
   3. For staining, wash the cells with 1 mL of prewarmed 1x PBS and subsequently fix the hiPSCs with 4% paraformaldehyde for 10 min at room temperature. Wash the fixed hiPSCs with 1x PBS and block unspecific binding sites with 200 µL of preincubation solution per well for 1 h at room temperature. Dilute primary antibodies Ki67 (1:300), OCT3/4 (1:200), SSEA4 (1:100) in antibody diluent.
   4. Clean a plastic foil with 70% ethanol and place it in a staining chamber filled with a water reservoir. Place droplets of 30 µL primary antibody dilutions on the foil. Place the fixed samples upside down in the dilution and incubate overnight at 4 °C.
   5. The next day, wash the samples three times with 1x PBS for 5 min and place 30 µL droplets of secondary antibody dilutions (1:1,000) on clean spots on the foil. Place the cover slides upside down in the dilution. Incubate for 1 h at room temperature in the dark.
   6. After incubation, wash three times with 1x PBS for 5 min. Mount the samples on glass slides in the mounting medium containing DAPI. Allow to dry overnight at room temperature and in the dark and image the samples using a confocal microscope (Figure 4).
3. Exclusion of bacteria and mycoplasma contamination
   1. To exclude bacteria contamination, transfer 500 µL of dissociated hiPSCs to 5 mL of prewarmed Luria-Bertani (LB) medium. See steps 5.4 to 5.6 for dissociation of the hiPSCs. Incubate overnight at 37 °C. If the LB medium appears turbid, bacteria contamination has likely occurred. Discard the cells.
   2. To exclude mycoplasma contamination, collect medium that has not been replaced for 2 days from 6 wells with around 90% confluent hiPSCs. Use quantitative polymerase chain reaction (qPCR) to detect mycoplasma contamination. Many commercial mycoplasma detection kits are available for this purpose.

8. Differentiation of hiPSCs into podocytes

1. Preparation and storage of growth factors
   1. To prepare a stock concentration of 10 mM Y27632, reconstitute 10 mg of Y27632 (molecular weight of 320.26 g/mol) in 3.12 mL of sterile water. Store 100 µL aliquots at -20 °C and dilute 1:1,000 in cell culture medium to reach a final concentration of 10 µM.
   2. To prepare a stock concentration of 30 mM CHIR99021, reconstitute 5 mg of CHIR99021 (molecular weight of 465.34 g/mol) in 358.2 µL of sterile DMSO. Store 50 µL aliquots at -20 °C and dilute 1:10,000 in cell culture medium to reach a final concentration of 3 µM.
   3. To prepare a stock concentration of 100 µg/mL activin A, reconstitute 100 µg of activin A in 1 mL of 1x PBS containing 0.1% bovine serum albumin (BSA). Store 100 µL aliquots at -20 °C and dilute 1:2,000 in cell culture medium to reach a final concentration of 50 ng/mL.
   4. To prepare a stock concentration of 100 µg/mL bone morphogenic protein 7 (BMP7), reconstitute 100 µg of BMP7 in 1 mL of sterile water containing 0.1% BSA. Store 100 µL aliquots at -20 °C and dilute 1:2,000 in cell culture medium to reach a final concentration of 50 ng/mL.
   5. To prepare a stock concentration of 100 µg/mL vascular endothelial growth factor (VEGF), reconstitute 100 µg of VEGF in 1 mL of sterile water. Store 100 µL aliquots at -20 °C and dilute 1:4,000 in cell culture medium to reach a final concentration of 25 ng/mL.
   6. To prepare a 10 mM stock concentration of all-trans retinoic acid, reconstitute 10 mg of all-trans retinoic acid in 3.33 mL of sterile DMSO. Store 100 µL aliquots at -20 °C and dilute 1:100 in cell culture medium to reach a final concentration of 0.5 µM.
2. Activation of WNT signaling pathway to induce mesoderm lineage
   1. For differentiation of hiPSCs into hiPSC-derived podocytes, coat cell culture plates or flasks suitable for hiPSC culture with coating solution for 1 h at 37 °C and 5% CO₂. The total volume of coating solution is 1 mL per 6-well culture plate or 4 mL per 10 cm cell culture plate.
   2. Aspirate the medium and wash the hiPSCs with prewarmed 1x PBS. Aspirate the PBS and add enzymatic cell detachment solution containing 10 µM Y27632 at a total volume of 1 mL per 6-well culture plate or 4 mL per 10 cm cell culture plate.
   3. Place the plate back in the incubator and incubate for 4 min at 37 °C and 5% CO₂. Rinse the hiPSCs off the plate using a 1,000 µL pipette. Transfer the detached cells to a conical tube.
   4. Wash the plate with fresh, prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632. Pool the washing medium in the conical tube to neutralize the dissociation reagent. Centrifuge at 200 x g for 5 min at 20 °C. Aspirate the supernatant and resuspend the cell pellet with 2 mL of fresh, prewarmed hiPSC culture medium containing 10 µM ROCK inhibitor Y27632.
   5. Count the cells and seed 1 x 10⁴ hiPSCs/cm². Distribute the cells by agitating three times in all directions. Let the hiPSCs attach overnight at 37 °C with 5% CO₂ without disturbance.
   6. The next day, replace the medium with 2 mL of prewarmed mesoderm differentiation medium containing 1x B27 supplement, 1% penicillin-streptomycin, 3 µM CHIR99021, 50 ng/mL activin A and 10 µM ROCK inhibitor Y27632.
3. Differentiation into nephron progenitor cells
   1. After 2 days, change the mesoderm medium to 2 mL of prewarmed nephron progenitor differentiation medium containing 1x B27 supplement, 1% penicillin-streptomycin, 3 µM CHIR99021, 50 ng/mL activin A, and 50 ng/mL BMP7 per 6-well culture plate or 6 mL per 10 cm cell culture plate. Change the medium every other day for the next 14 days.
      NOTE: Nephron progenitor cells proliferate and can be passaged and frozen after 7 days of differentiation in nephron progenitor expansion medium.
   2. To split the nephron progenitor cells, coat the cell culture plastics suitable for hiPSC culture with coating solution for 1 h at 37 °C. Replace the coating solution with nephron progenitor expansion medium. Aspirate the media and wash the cells with prewarmed 1x PBS. Remove the PBS and dissociate the cells with 1x trypsin-EDTA.
   3. Incubate for 5 min at 37 °C and 5% CO₂. Monitor detachment of the cells using a phase contrast microscope. If necessary, tap against the side of the plastic to detach the cells from the surface. Rinse the cells off the plate and transfer to a conical tube. Wash the empty flask or plate with the same volume of prewarmed nephron progenitor expansion medium as the dissociation reagent.
   4. Pool the washing medium in the conical tube. Centrifuge at 200 x g for 5 min at 20 °C. Aspirate the supernatant and resuspend the cell pellet with 2 mL of fresh, prewarmed nephron progenitor expansion medium. Count the cells and seed 1.5 x 10⁴ nephron progenitor cells per cm² for further differentiation.
      NOTE: To freeze the nephron progenitor cells, resuspend 1 x 10⁶ cells in 1 mL of cold serum-free cryopreservation medium and transfer to a cryovial. Freeze in a freezing container at -80 °C overnight and transfer to a liquid nitrogen tank for long-term storage.
   5. Place the plates back in the incubator and distribute the cells evenly by agitation three times in all directions. Change the medium the next day to fresh, prewarmed nephron progenitor differentiation medium. Replace the medium every other day until the cells are differentiated for 14 days in nephron progenitor differentiation medium.
4. Terminal differentiation into hiPSC-derived podocytes
   1. Aspirate the medium and add 2 mL of prewarmed podocyte differentiation medium containing 1x B27 supplement, 1% penicillin-streptomycin, 3 µM CHIR99021, 50 ng/mL activin A, 50 ng/mL BMP7, 25 ng/µL VEGF, and 0.5 µM all-trans retinoic acid per 6-well culture plate or 6 mL per 10 cm cell culture plate. Place the plates back in the incubator at 37 °C and 5% CO₂. Change the medium every other day for 4 days with fresh, prewarmed podocyte differentiation medium.
   2. To keep the hiPSC-podocytes in culture after differentiation, feed the cells twice a week with podocyte maintenance medium containing 10% fetal calf serum, 1% penicillin-streptomycin, and 0.1% insulin-transferrin-selenium.
      ​NOTE: Terminal differentiated hiPSC-podocytes do not proliferate anymore. However, it is possible to keep them in cell culture for up to 4 weeks.
5. Thawing and expansion of frozen nephron progenitor cells
   1. Coat a new cell culture plastic flask suitable for hiPSC culture with coating solution for 1 h at 37 °C and 5% CO₂. Replace the coating solution with 5 mL of nephron progenitor expansion medium.
   2. Prepare a 15 mL conical tube with 7 mL of prewarmed nephron progenitor expansion medium. Thaw the frozen cells at 37 °C in a water bath without movement for approximately 20 s. Take the cryovial out of the water bath when half of the cells are thawed, and some ice is remaining.
   3. Spray the cryovial with 70% ethanol and place in a biosafety cabinet. Transfer thawed cells to the conical tube with the prewarmed nephron progenitor expansion medium. Use a 1,000 µL pipette and let the cells run down the wall of the tube very slowly.
   4. Wash the empty cryovial with 1 mL of nephron progenitor expansion medium to collect the remaining cells and pool in the conical tube. Centrifuge at 200 x g for 5 min at 20 °C and resuspend the cell pellet in fresh, prewarmed nephron progenitor expansion medium.
   5. Transfer the thawed cells to the coated flask and change the medium the next day to fresh, prewarmed nephron progenitor expansion medium. Before further differentiation, let the cells proliferate in nephron progenitor expansion medium by changing the medium every other day, and proceed from step 8.3.5.

9. Characterization of hiPSC-derived podocytes by immunofluorescence staining

1. To check the differentiated podocytes for the podocyte-specific marker via immunofluorescence staining, place plastic coverslips in a 24-well plate and coat with 250 µL of coating solution for 1 h at 37 °C and 5% CO2. Replace the coating solution with 1 mL of prewarmed nephron progenitor expansion medium. Seed 2.5 x 104 dissociated nephron progenitor cells per 24-well plate. See steps 8.3.2 to 8.3.4 for dissociation of nephron progenitor cells.
2. Let the cells attach overnight at 37 °C and 5% CO2 without disturbance. Replace the medium the next day with prewarmed nephron progenitor differentiation medium.
3. Finish differentiation by changing the medium every other day until the cells are differentiated for a total of 14 days in nephron progenitor differentiation medium and an additional 5 days in podocyte differentiation medium.
4. After the final differentiation, wash the cells with 1 mL of prewarmed 1x PBS. Fix the hiPSC-podocytes with 4% paraformaldehyde for 10 min at room temperature. Wash the fixed cells with 1x PBS and block unspecific binding sites with 200 µL of preincubation solution per well for 1 h at room temperature.
5. Dilute the primary antibodies synaptopodin (1:200), podocin (1:100), and nephrin (1:25) in antibody diluent and place droplets of 30 µL primary antibody dilution on a clean plastic foil in a staining chamber containing a water reservoir.
6. Place cover slides with fixed hiPSC-podocytes upside down in the dilution. Incubate overnight at 4 °C. The next day, wash three times with 1x PBS for 5 min. Dilute the secondary antibodies (1:1,000) in 1x PBS and place droplets of 30 µL secondary antibody dilution on clean spots on the foil. Place the cover slides upside down in the dilution.
7. Incubate for 1 h at room temperature in the dark. After incubation, wash three times with 1x PBS for 5 min. Mount the samples on glass slides in mounting medium containing DAPI. Allow to dry overnight at room temperature and in the dark. Image the samples using a confocal microscope.