human iPSC-derived Sox9+ sclerotome-like cells , biol rep 3
Homo sapiens
source_name
UC01
protocol
Both the human iPSC-derived Sox9+ sclerotome-like cells and human iPSC-derived Sox9- cells were obtained by differentiating the day-0 UC01 iPSCs (Sox9-IRES-tdTomato) to day-6 cells stepwise and sorting out the Sox9+ and Sox9- cells , respectively. All differentiation was conducted in chemically defined medium with insulin (CDMi) supplemented with indicated proteins and chemical molecules. To prepare CDMi, 50% IMDM (Gibco, Cat# 31980-097) and 50% F12 (Gibco, Cat# 31765-092) were mixed with 1% v/v concentrated lipids (Gibco, Cat# 11905-031), 450 μM monothioglycerol (Sigma, Cat# M6145), 0.7 μg/mL insulin (Solarbio, Cat# I8830), 15 μg/ml transferrin (Sigma, Cat# T0665), 1% v/v penicillin/streptomycin (Gibco, Cat# T0665), and 1 mg/mL polyvinyl alcohol (Sigma, Cat# P8136-250G). The Polyvinyl alcohol was first solved at 25 mg/ml in ultrapure water (UP H2O) by heating to 85 ℃ and then cooled on ice before addition to the medium. To start differentiation, day-0 UC01 iPSCs (Sox9-IRES-tdTomato) were gently washed with the wash medium (WM) containing 3 mg/ml bovine serum albumin (BSA) in DMEM/F12 and induced to primitive streak (PS) by CDMi containing Activin A (30 ng/ml, Solarbio, Cat#P00101), CHIR99021 (7 μM, MCE, Cat#HY-10182) and bFGF (20 ng/ml, Peprotech, Cat#100-18B) for 24 hours; the next day, day-1 PS was washed with WM and differentiated to presomitic mesoderm (PSM) by CDMi containing SB431542 (20 μM, MCE, Cat#HY-10431), CHIR99021 (3 μΜ), LDN193189 (125 nM, MCE, Cat#HY-12071A), bFGF (40 ng/ml) for another 24 hours; subsequently, the day-2 PSM was with WM and differentiated to somite (SM) in CDMi containing XAV939 (0.5 μM) and PD0325901 (1 μM, MCE, Cat#HY-10254) for 24 hours; the following day, the SM was washed with WM, and was induced to day-6 cells by CDMi containing SAG (200 nM) and LDN193189 (600nM) for 72 hours with medium changed every day.
cell line
UC01
cell type
induced pluripotent stem cell-derived cells
genotype
Sox9-IRES-tdTomato
treatment
sclerotome differentiation