Uncoated petri dishes

mESCs were trypsinized to obtain a single mESC suspension, which was then transferred to uncoated Petri dishes (SPL Lifescience, Pocheon, Korea). The cells were cultured for 3 days in differentiation medium (high-glucose, glutamine-free Dulbecco’s modified Eagle’s medium) supplemented with 15% ES-qualified fetal bovine serum (HyClone, Logan, UT, USA), 2 mM GlutaMAX, 0.1 mM non-essential amino acid stock, 0.1 mM β-mercaptoethanol, 100 U ml⁻¹ penicillin and 100 U ml⁻¹ streptomycin).

Human iPSCs were differentiated into MSCs as previously described (Choi et al., 2017b ; Mahmood et al., 2010 (link)). Briefly, human iPSC colonies were dissected into small clumps (approximately 0.5 mm × 0.5 mm in size). These clumps were transferred to uncoated Petri dishes (SPL Lifesciences, Pocheon, Korea) and incubated in EB medium for 2 days. The EB medium consisted of DMEM/F12 (Invitrogen, Carlsbad, CA) with 10% Knockout SR (Invitrogen), 1% nonessential amino acids (Invitrogen), 1.2 mg/mL sodium carbohydrate, and 0.1 mM β-mercaptoethanol (Sigma, St. Louis, MO). In this medium, the clumps spontaneously aggregated to form embryoid bodies (EBs). These EBs were cultured in the same medium supplemented with 10 μM SB431542 (Cayman Chemical, Ann Arbor, MI) for 8 d with media changes every other day. After 10 d of incubation, the EBs were attached to fibronectin-coated dishes (BD Biosciences, Franklin Lakes, NJ) and then further cultured in DMEM/F12 supplemented with 1 μM SB431542, 1% insulin plus transferrin liquid supplement (Sigma), 2% B27 supplement (Invitrogen), and 1% CD lipid (Invitrogen) for 8–10 additional days with medium changes every other day. For MSC maturation, the cells were then cultured in α-MEM (Invitrogen) containing 10% FBS for 14 days with medium changes once every 3 days.