Work with iPS and ES cells was conducted under the approval and auspices of the University of Washington Embryonic Stem Cell Research Oversight Committee. Specific cell lines used in this study are described below and were sourced from commercially available iPS and ES cell lines obtained with informed consent. Stem cell stocks were maintained in mTeSR1 media with daily media changes and passaging using Accutase (STEMCELL Technologies). One thousand to six thousand cells per well were placed in each 24-well plate precoated with 300 μL of DMEM-F12 containing 0.2 mg/mL Matrigel and sandwiched the following day with 0.2 mg/mL Matrigel in mTeSR1 (STEMCELL Technologies) to produce scattered, isolated spheroid colonies. Forty-eight hours after sandwiching, spheroids were treated with 12 μM CHIR99021 (Tocris Bioscience) for 36 hours, then changed to RB (Advanced RPMI + 1× Glutamax + 1× B27 Supplement, all from Thermo Fisher Scientific) and replaced every 3 days thereafter. Organoids were differentiated for 21 days from the time of plating, at which time tubular structures had formed. Gene-edited PKD2−/− organoids were picked from the adherent plate at day 21, placed in suspension culture with RB media replaced every 3 days until day 30 when cyst growth was prominent (37 (link)).
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