Stemfit basic02

The healthy control iPSC line known as wild type C (WTC) used in these studies is an extensively characterized line from a healthy individual (Kreitzer et al., 2013 (link)) that is the parental line for the Allen Institute Cell Collection¹. A CMT2E iPSC line derived from a female individual with the N98S mutation in NEFL causing childhood onset CMT has been previously characterized (Saporta et al., 2015 (link); Maciel et al., 2020 (link)). The N98S mutation occurs in the Coil1A domain of the neurofilament light chain (NF-L) protein at the 98th amino acid in both human and mouse orthologs. The mutation has also been referred to in the literature as N97S, likely due to variable inclusion of the start codon in the numbering system. iPSCs were cultured in Stemfit Basic02 (Ajinomoto) on plates coated with matrigel (Corning, 356231) at 37°C, 5% CO₂, 85% humidity. iPSC cultures were passaged every 3–4 days with Accutase (Stem Cell Technologies, 07920) or ReLeSR (Stem Cell Technologies, 05872) into Stemfit supplemented with 10 μm Y-27632 (SelleckChem, S1049).

H9 (WiCell) human embryonic stem cells and TCiPS1 induced pluripotent stem cells were maintained on hESC-qualified Geltrex (Thermo Fisher Scientific) coated plates in feeder-free culture using StemFit ^® Basic02 (Ajinomoto Co., Inc.) supplemented with 10 ng/mL of FGF2 (Peprotech), as previously reported (Morizane and Bonventre, 2017a (link)). The hPSC line was passaged weekly, using Accutase (STEMCELL technologies) for dissociation and Y27632 (Tocris) to facilitate adhesion on passaging. H9 passage numbers 50–62 and TCiPS1 passage numbers 18–30 were used for all experiments.

H9 human embryonic stem cells (ESCs; WiCell) and BJFF.6 human induced PSCs (provided by Prof. Sanjay Jain at Washington University) were maintained in StemFit Basic02 (Ajinomoto Co., Inc.) supplemented with 10 ng/ml FGF2 (Peprotech) as previously described (Morizane et al., 2015 (link); Morizane and Bonventre, 2017 (link)). Stem cells at passages 34–55 were used for the experiment. Kidney organoids were generated using a previously reported protocol (Morizane et al., 2015 (link); Morizane and Bonventre, 2017 (link)). Briefly, hPSCs were differentiated into SIX2⁺ nephron progenitor cells (NPCs) in 2D culture with 80–90% efficiency. The NPCs were later transferred to 3D cultures in 96-well ultra-low attachment plates (Corning) and differentiated into nephron organoids. Organoids at d21-49 were used for functional assays.