title: Mesangial lineage cell, iPSC derived, Normoxia, rep1
organism: Homo sapiens
description: hiPSCs were first treated using an enzymatic method with a 1:1 mixture of TrypLE Select Enzyme and 0.5 mM EDTA/PBS for 5 min and washed with PBS(-). The cells were then detached using a cell scraper, dissociated into single cells by gentle pipetting and seeded onto 24-well plates (CORNING) at a density of 1.3x10^4 cells/cm^2 with 0.5 mL of Stem Fit AK02N supplemented with 10 uM Y-27632 (day -1) and 2.4 uL/mL iMatrix-511 (Nippi). After 24 h (day 0), the cells were cultured with serum-free differentiation medium consisting of DMEM/F12 Glutamax medium (Thermo Fisher Scientific), B27 supplement minus vitamin A (Thermo Fisher Scientific), and 0.5X Penicillin/streptomycin (hereafter called basal medium) supplemented with 5 uM CHIR99021 (Wako), 10 nM RA (SIGMA), 1 ng/mL BMP4 (Peprotech), and 100 ng/mL bFGF (Wako). After another 24 h (day 1), the cells were cultured with 5 uM CHIR99021, 100 ng/mL bFGF, and 100 nM LDN (_AxonMedchem). After a third 24 h (day 2), the medium was switched to basal medium containing 5 uM CHIR99021, 1 ng/mL BMP7, and 10 uM A83-01 (Wako). After a fourth 24 h (day 3), the medium was changed to basal medium containing 5 uM CHIR99021, 30 ng/mL bFGF, 10 ng/mL activin A (R&D Systems), and 30 uM Y-27632 (Wako). After a fifth 24 h (day 4), the cells were washed with PBS (-), treated with Accumax (Innovative Cell Technologies, Inc.), dissociated into single cells by gentle pipetting. On day 4 of PPS induction, the dissociated cells were cryopreserved using STEM-CELLBANKER (ZNQ). The thawed cells were seeded on 96-well U-bottom plates (Sumitomo Bakelite or Thermo Fisher Scientific) at a density of 2.0x10^4 cells/well with basal medium containing 5 uM CHIR99021, 30 ng/mL bFGF, 10 ng/mL activin A and 30 uM Y-27632, to form cellular aggregates and cultured for 48 h. On day 6, the cells were incubated with basal medium containing 1 uM CHIR99021, 0.1 uM RA, 500 nM Smoothened Agonist (SAG) (Selleck), 10 uM SB431542 (Nakalai tesque) and 10 ng/mL IL-1b (Wako). On day 8, the medium was changed to basal medium containing 1 uM CHIR99021, 0.1 uM RA, 500 nM/mL SAG and 10 uM SB431542 for 72 h. For mesangial lineage induction, on day 11, the cells were transferred to low cell binding 24 well plate (Sumitomo Bakelite) and incubated with basal medium containing 1 uM CHIR99021, 0.1 uM RA, 100 ng/mL BMP7, 10 uM SB431542 and 100 ng/mL VEGF (Peprotech) for 96 h under normoxic condition.
attributes.tag: sample_name
attributes.value: 0716MES_S2
attributes.tag: age
attributes.value: 36
attributes.tag: biomaterial_provider
attributes.value: Shinya Yamanaka
attributes.tag: cell_line
attributes.value: 201B7
attributes.tag: collection_date
attributes.value: 2022-07-16
attributes.tag: geo_loc_name
attributes.value: Japan:Kyoto
attributes.tag: isolate
attributes.value: 201B7
attributes.tag: sex
attributes.value: female
attributes.tag: tissue
attributes.value: dermal fibroblast
attributes.tag: BioSampleModel
attributes.value: Human