Deriving Human Kidney Podocytes from Induced Pluripotent Stem Cells: A Procedure for Directed Differentiation of Mature Kidney Podocytes from Stem Cells

1. Preparation of Reagents

1. Dilute thawed 5x hiPS cell culture media (CCM) supplement in hiPS cell culture basal medium to obtain a 1x solution of hiPS CCM.
   NOTE: Frozen 5x hiPS CCM supplement requires a slow thawing process, ideally in 4 ˚C for overnight. Aliquots of the 1x hiPS CCM can be stored for up to 6 months at -20 ˚C.
2. Preparation of basement membrane (BM) matrix 1-coated plates for hiPS cell culture: Thaw BM matrix 1 overnight on ice at 4 ˚C. Once thawed, prepare aliquots with appropriate dilution factors as suggested by the manufacturer.
   NOTE: Typically, aliquots are prepared for subsequent dilution in 25 mL of cold DMEM/F12 in a 50 mL conical tube followed by thorough mixing to completely dissolve the BM matrix 1 and avoid the formation of residual crystals.
3. Transfer 1 mL of the BM matrix 1 solution to each well of a 6-well plate and incubate at 37 ˚C for 1–2 h or at 4 ˚C for a minimum of 24 h, wrapped in paraffin film. The BM matrix 1-coated plates can be stored at 4 ˚C for up to 2 weeks.
4. Preparation of Basement membrane (BM) matrix 2-coated plates: Dilute appropriate amounts of BM matrix 2 in 9 mL sterile distilled water to prepare a final concentration of 5 µg/mL. Add 700 µL of the BM matrix 2 solution to each well of a 12-well plate and incubate the plate at room temperature for 2 h or overnight at 4 ˚C.
5. Prepare 100 µg/mL stock solutions each of BMP7, Activin A, and VEGF as follows: Reconstitute BMP7 in sterile distilled water containing 0.1% (wt/vol) BSA and reconstitute Activin A and VEGF separately in sterile PBS containing 0.1% (wt/vol) BSA. To avoid frequent freeze-thaw cycles, prepare 100 µL aliquots from each stock solution and store at -20 ˚C for up to 6 months.
6. Prepare a 10 mM stock solution of Y27632 by dissolving 10 mg of Y27632 in 3.079 mL of sterile distilled water. Aliquot 100 µL from the stock and store at -20 ˚C for up to 6 months.
7. Dissolve 2 mg of CHIR99021 in 143.4 µL of sterile DMSO to prepare a 30 mM stock solution. Prepare 5 µL aliquots and store at -20 ˚C for up to 1 month (or according to the manufacturer’s recommendation).
8. Dissolve 10 mg of all-trans retinoic acid in 3.33 mL sterile DMSO. Prepare 500 µL aliquots and store at -20 ˚C for up to 6 months.

2. Preparation of Culture Media

1. Prepare the mesoderm differentiation medium by reconstituting corresponding stock solutions to a final concentration of 100 ng/mL Activin A, 3 µM CHIR99021, 10 µM Y27632, and 1x B27 serum-free supplement in an appropriate volume of DMEM/12 with glutamine supplement.
   NOTE: The mesoderm differentiation medium should be freshly prepared before differentiation steps and at a volume appropriate for the scale of the experiment (typically, 50 mL of medium is adequate for two 12-well plates).
2. Prepare intermediate mesoderm differentiation medium by reconstituting corresponding stock solutions to a final concentration of 100 ng/mL BMP7, 3 µM CHIR99021, and 1x B27 serum-free supplement in DMEM/F12 with a glutamine supplement.
   NOTE: If needed, the medium can be supplemented with 1% (vol/vol) Penicillin-Streptomycin. Adjust the volume of the medium by using DMEM/F12 with glutamine supplement. This medium can be prepared in large batches; however, it is recommended to store in smaller aliquots (e.g., 45 mL in 50 mL conical tubes) to avoid repeated freeze-thaw cycles. This media can be stored at -20 ˚C for up to 3 months and can be thawed at 4 ˚C overnight prior to use.
3. Prepare the podocyte induction medium by reconstituting to a final concentration 100 ng/mL BMP7, 100 ng/mL activin A, 50 ng/mL VEGF, 3 µM CHIR99021, 1x B27 serum-free supplement, and 0.1 µM all-trans retinoic acid in DMEM/F12 with glutamine supplement. Protect medium from light (e.g., by wrapping container with foil paper).
   NOTE: This medium can be prepared in large batches and stored in the dark at -20 ˚C for up to 3 months. Frozen aliquots should be thawed overnight at 4 ˚C prior to use.
4. Prepare 25 mL of trypsin neutralizing solution by adding 10% (vol/vol) heat-inactivated FBS in DMEM/F12 and filter under sterile conditions.
5. For post-differentiation maintenance of the stem cell-derived podocytes, prepare Complete Medium with podocyte maintenance media by adding the supplement to the basal medium as per the manufacturer’s guidelines, and store at 4 ˚C for up to two weeks.

3. Feeder-free hiPS Cell Culture Using hiPS Cell Culture Medium

1. Aspirate the residual solution of BM matrix 1 from the pre-coated plates and wash the wells 3 times with 1 to 2 mL of warmed DMEM/F12.
2. Aspirate spent hiPS CCM from the hiPS cells and rinse the cells 3 times with warmed DMEM/F12. Add 1 mL of warm cell detachment solution and incubate for 1 min at 37 ˚C to help dissociate the cells. Perform visual inspection of cells under a tissue culture microscope and ensure that the edges of the cell colonies appear rounded, then quickly aspirate the cell detachment solution from the cells (ensuring that the cell colonies are still attached to the plate, albeit loosely). Gently rinse cells once with DMEM/F12 to remove the cell detachment solution.
3. Add 3 mL of hiPS CCM to the hiPS cells (in each well of a 6-well plate) that were treated with the cell detachment solution. Scrape colonies by using a cell lifter, and gently pipette cell suspension up and down to dislodge the loosely adhered cells. Wash the plate thoroughly to ensure all the cells are harvested.
   NOTE: Use of a 5 mL pipette is recommended to avoid excess shear on the cells.
4. Transfer 0.5 mL of the cell suspension into each well of a new BM matrix 1-coated 6-well plate containing 2 mL of hiPS CCM per well. Move the plate in figure-eight fashion to distribute cell colonies within wells and incubate at 37 ˚C in a 5 % CO₂ incubator. Refresh medium daily until the cells are ready to be passaged or used for the differentiation experiment (approximately 70 % confluency).
   NOTE: The ideal colony size for routine passaging of iPSCs is between 200-500 µm under feeder-free conditions using hiPS CCM (without ROCK inhibitor). If cells are individualized during treatment with dissociation enzymes or buffers, the hiPS CCM can be supplemented with ROCK inhibitor (e.g., 10 µM Y27632) to improve cell viability.

4. Differentiation of hiPS Cells into Mesoderm Cells (Days 0–2)

1. While the hiPS cell cultures are in the exponential growth phase (approximately within 4 days of culture after passaging, and around 70% confluency), visually inspect for the presence of spontaneously differentiated cells within and around the edges of the colonies. If necessary, aseptically scrape off areas of differentiation.
2. Aspirate hiPS CCM from hiPS cells and rinse the cells 3 times with warm DMEM/F12. Incubate the cells with 1 mL of enzyme-free cell dissociation buffer for 10 min at 37 ˚C and check for the dissociation under a microscope. Due to inherent differences between different hiPS cell lines, the actual incubation time for the cell dissociation buffer must be determined for a given cell line.
3. Gently scrape the well with a cell lifter to dislodge loosely adhered cells and transfer the cell suspension to a 15 mL conical tube, followed by pipetting up and down several times to individualize the hiPS cells.
   1. Bring cell suspension to the 15 mL volume with warm DMEM/F12 and centrifuge for 5 min at 290 x g at room temperature.
   2. Gently aspirate the supernatant and resuspend the cells with warm DMEM/F12 for another round of centrifugation to remove residual BM matrix 1 and dissociation buffer components.
4. Aspirate the supernatant and resuspend cells in 1 mL of mesoderm induction medium as described above. Count the total number of cells using a hemocytometer or coulter counter to determine the appropriate volume of mesoderm differentiation medium necessary to achieve a concentration of 1 x 10⁵ cells/mL.
5. Aspirate ECM solution from the BM matrix 2-coated plates and rinse the plates twice with warm DMEM/F12. Mix the hiPS cell suspension gently by pipetting a few times. Transfer 1 mL of the cell suspension to each well of the BM matrix 2-coated 12-well plates and then gently shake the plates to distribute the cells more evenly.
6. Incubate the plate at 37 ˚C in a 5% CO₂ incubator. Refresh the mesoderm induction medium the next day.
   NOTE: After 2 days, hiPS cell-derived mesoderm cells would be ready for intermediate mesoderm induction.

5. Differentiation of hiPS Cell-derived Mesoderm Cells into Intermediate Mesoderm (Days 2–16)

1. On day 2 of the differentiation protocol, aspirate mesoderm induction medium and replenish with 1 mL per well intermediate mesoderm induction medium.
2. Refresh medium every day to maintain an accurate threshold of growth factors and small molecules for the metabolically active cells. If there is substantial cell growth and rapid depletion of media nutrients (indicated by the yellowing of the media), the volume of the intermediate mesoderm differentiation medium can be increased to 1.3 mL per well of the 12-well plates.
3. Culture cells for additional 14 days to obtain intermediate mesoderm cells. By day 16, these cells can be cryopreserved for later use.

6. Differentiation of hiPS Cell-derived Intermediate Mesoderm Cells into Podocytes (Days 16–21)

1. Rinse the intermediate mesoderm cells with warm DMEM/F12 followed by incubation of the cells with 0.5 mL per well of 0.05 % trypsin-EDTA for 3 min at 37 ˚C. Perform visual inspection to ensure that the cells are beginning to dissociate.
2. Scrape cells using a cell lifter and pipette the cell suspension several times using a 1,000 µL pipette tip to obtain individualized (or small clumps of) cells.
   NOTE: At this stage, ensure that the cells are fully dissociated as aggregated cells may fail to acquire a terminally differentiated phenotype within the timeline of the protocol.
3. Add about 2 mL per well of trypsin neutralizing solution to stop the activity of trypsin.
4. Transfer cells to a 50 mL conical tube and bring the volume up to 50 mL using DMEM/F12, and then centrifuge the cell suspension for 5 min at 201 x g at room temperature.
5. Aspirate the supernatant and resuspend cells in the podocyte induction medium. For optimal results, ensure a final seeding density of approximately 100,000 cells/well of a 12-well plate. Add the cell suspension to the BM matrix 2-coated plates and gently shake the plate to help distribute the cells more evenly.
6. Incubate cells at 37 ˚C and 5% CO₂ and refresh medium daily for up to 5 days to obtain podocytes by day 21.
   NOTE: The resulting hiPS cell-derived podocytes can be maintained in culture for 2–4 additional weeks by using complete medium with podocyte maintenance media. Once in podocyte maintenance media, the cells can be fed every other day and may be used for subsequent studies or downstream analyses.