SAMD00612368
External Id
Osafune Laboratory, Kyoto University, Center for iPS Cell Research and Application (CiRA)
INSDC center name
2024-01-18T00:00:00Z
INSDC first public
2024-06-02T16:08:17.707Z
INSDC last update
DRS360133
INSDC secondary accession
live
INSDC status
Human
NCBI submission model
Human.1.0
NCBI submission package
DRS360133
SRA accession
36
age
Shinya Yamanaka
biomaterial_provider
201B7
cell_line
2022-07-24
collection_date
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.
description
Japan:Kyoto
geo_loc_name
201B7
isolate
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
0724BMES_S4
sample_name
female
sex
dermal fibroblast
tissue
Mesangial lineage cell, iPSC derived, Normoxia, rep2
title