Chir99021

hiPS cells were first dissociated from Matrigel-coated plates by treatment with StemPro Accutase (Thermo Fisher Scientific) and centrifuged twice at 1200 rpm for 5 min each in DMEM/F12. The cells were seeded on laminin 511-E8-coated plates with a mesoderm differentiation medium consisting of DMEM/F12 with GlutaMax (GIBCO) supplemented with 100 ng/mL Activin A (Thermo Fisher Scientific), 3 μM CHIR99021 (Stemgent), 10 μM Y27632 (TOCRIS), and 1X concentration of B27 serum-free supplement (GIBCO). After 2 days of differentiation, the cells were incubated for a minimum of 14 days with an intermediate mesoderm induction medium consisting of DMEM/F12 with GlutaMax supplemented with 100 ng/mL BMP7 (Thermo Fisher Scientific), 3 μM CHIR99021, and 1X concentration of B27 serum-free supplement. To induce podocyte phenotype, the intermediate mesoderm cells were dissociated by treatment with 0.05% Trypsin-EDTA and cultured at 1:4 splitting density on freshly prepared laminin 511-E8-coated plates, and fed daily for 4–5 days with a podocyte induction medium consisting of DMEM/F12 with GlutaMax supplemented with 100 ng/mL BMP7, 100 ng/mL ActivinA, 50 ng/mL VEGF (Thermo Fisher Scientific), 3 μM CHIR99021, 1X concentration of B27 serum-free supplement, and 0.1 μM all-trans retinoic acid (Stem Cell Technologies).

Human HeLa cells and mouse embryonic stem cells (mESC) were purchased from ATCC. HeLa cells were grown in DMEM (Gibco, 11965092) media supplemented with 10% FBS (Gibco) and 1% 100× Pen/Strep (Gibco). WT, control knockout, and Mettl3 conditional knockout (cKO) mESCs were maintained in DMEM (Invitrogen) supplemented with 15% FBS (Gibco), 1% nucleosides (100×) (Millipore), 1 mM L-glutamine (Gibco), 1% nonessential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 1,000 U/ml LIF (Millipore), 3 μM CHIR99021 (Stemcell), and 1 μM PD0325901 (Stemcell). All cells were cultured at 37 °C under 5.0% CO₂.
Mettl3 cKO mES cell lines were generated following previously reported methods⁵³ . Briefly, mESCs derived from Mettl3^flox/flox mouse blastocyst were transfected with 200 ng PB-CAG-Puromycin-P2A-CreERT2 and 100 ng PBase by electroporation. After 24 h, electroporated cells were treated with 1 μg/ml Puromycin to generate stable Mettl3^flox/flox; CreERT2 mES clones. To induce deletion, Mettl3^flox/flox; CreERT2 ESC cells were treated with 1 μg/ml 4-hydroxytamoxifen (Sigma). These Mettl3 KO cells were cultured for 48 h before harvesting. Untreated Mettl3^flox/flox; CreERT2 ESC cells were used as ctrl mESCs.

iPSCs were differentiated into kidney organoids following the previously published protocol by Freedman et al. (5 (link)) (Figure 1B). Briefly, iPSCs were dissociated with 1:3 Accutase and plated onto 24-well plates precoated with 0.5% GelTrex in mTeSR1 supplemented with 10 μM Y-27632 ROCK Inhibitor (STEMCELL Technologies). After 24 hours, another layer of GelTrex at 1.5% was added in mTeSR1 media. At the end of day 4, the medium was replaced with Advanced RPMI (Gibco) supplemented with 12 μM CHIR-99021 and 10 ng/ml noggin (STEMCELL Technologies). Approximately 60 hours later, the medium was changed to Advanced RPMI with B27 (Gibco). Organoids were cultured in this medium until collection at day 25.