Stemdiff apel2 medium

Kidney organoids were generated using a previously described protocol (37 (link)) with slight modifications. A total number of 3.75 × 10⁵ ThF iPSCs were plated in a T25 flask in the mTeSR1 medium with ROCK Inhibitor Y-27632 (10 μM, Stem cell Technologies, #72304). After 24 h, cells were treated with CHIR99021 (8 μM, R&D systems, #4423/10) in the STEMdiff APEL2 medium (Stem Cell Technologies, #05270) for 4 days, followed by recombinant human FGF-9 (200 ng/mL, Peprotech, #100-23) and heparin (1 μg/mL, Sigma-Aldrich, #H4784) for an additional 3 days. At day seven, cells were dissociated into single cells using AccutaseTM (Stem Cell Technologies, #07920). 5 × 10⁵ cells were pelleted at 350x g for 2 min and transferred onto a 6-well transwell membrane (Stem Cell Technologies, #3450). Pellets were incubated with CHIR99021 (5 μM) in the APEL2 medium for 1 h at 37°C. Then the medium was changed to the APEL2 medium with FGF-9 (200 ng/mL) and heparin (1 μg/mL) for an additional 5 days, and an additional 2 days with heparin (1 μg/mL). Medium was changed every other day. The organoids were maintained in APEL2 medium with no additional factors until day 25. Then kidney organoids were treated with PBS, PAN (150 μg/mL) with or without AC1903 (30 μM) for 72 h before the downstream experiments.

Human iPSC-derived podocytes (iPodos) were generated using the cited protocol with a few modifications (32 (link)). A total number of 3.75 × 10⁵ ThF human iPSCs were seeded in a Matrigel-coated T25 flask in mTeSR1 medium (Stem Cell Technologies, #85870) with ROCK inhibitor, Y-27632 (10 μM, Stem cell Technologies, #72304). After 24 h cells were treated with a 1:1 mixture of DMEM/F12 + GlutaMAX (Life Technologies, #10565-018) and Neurobasal media, supplemented with N2 and B27 (Life Technologies, #21103049), CP21R7 (1 μM, Cayman Chemical, #20573), and BMP4 (25 ng/mL, Peprotech, #AF-120-05ET), for 3 days. On day four, the medium was replaced with STEMdiff APEL2 medium (Stem Cell Technologies, #05270) supplemented with FGF9 (200 ng/mL, Peprotech, #100-23), BMP7 (50 ng/mL, Peprotech, #120-03), and Retinoic Acid (100 nM, Sigma-Aldrich, #R2625) for 2 days. On day six, cells were dissociated with Accutase (Stem Cell Technologies, #07920) and 2 × 10⁵ cells were seeded on Type I Collagen-coated 6-well dishes and cultured until day fourteen in DMEM/F12+GlutaMAX medium supplemented with 10% FBS (Life Technologies, #16140071). Vitamin D3 (100 nM, Tocris Bioscience, #4156), and Retinoic Acid (100 μM, Sigma-Aldrich, #R2625) were added every other day. Cells were fully differentiated and ready to use from Day 12 to Day 14.

As it was previously described by Xu et al. [27 (link)], iPS cells were passaged with 1 U/mL dispase (Thermo Fisher Scientific) and transferred to suspension culture in myogenesis-promoting medium (STEMdiff APEL 2 Medium, STEMCELL Technologies, Vancouver, BC, Canada), 10 ng/mL bFGF, 0.5 µM BIO (Sigma-Aldrich), 20 µM forskolin (Sigma-Aldrich), and 100 U/mL penicillin/streptomycin) for EB formation. The medium was changed every other day. After 7 days, cells were seeded on a dish coated with Matrigel (BD) in a 1:3 ratio in DMEM high glucose medium (Thermo Fisher Scientific) supplemented with 2% horse serum (Thermo Fisher Scientific) and 100 U/mL penicillin/streptomycin and cultured for further 35 days with daily medium change.

iPSCs were pre-treated with ROCK inhibitor for 1 h before single cell dissociation by Accutase^TM (Gibco). The HPC induction protocol was slightly modified from the protocol used by Lupo KB, et al. [22 (link)] by means of cell density and duration of each phase. Briefly, 5000 single cells were seeded in each well of an ultra-low attachment, round-bottom 96-well plate in 100 μL hematopoietic differentiation medium comprising STEMdiff APEL2 medium (STEMCELL Technologies), 40 ng/mL stem cell factor (SCF), 20 ng/mL bone morphogenic protein-4 (BMP-4), 20 ng/mL vascular endothelial growth factor (VEGF), and 10 μM ROCK inhibitor for the first 3 days. All cytokines used were obtained from R&D Systems (Minneapolis, MN, USA). The plate was centrifuged at 250× g for 5 min to promote EB formation and incubated for 6 days, during which the medium was changed every 3 days by removing 70 μL of medium from each well and adding 100 μL of freshly prepared medium without ROCK inhibitor.
On day 6, 30 EBs were transferred onto a Matrigel-coated 6-well plate in 4 mL NK cell differentiation medium consisting of STEMdiff APEL2 medium, 20 ng/mL SCF, 20 ng/mL IL-7, 10 ng/mL IL-15, and 10 ng/mL Flt3 ligand, which was supplemented with 5 ng/mL IL-3 for the first week. The medium was half-changed every two to three days for 3–4 weeks.