title: IPC, iPSC derived, rep3
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.
attributes.tag: sample_name
attributes.value: 0825SI_S13
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: 2022-08-25
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