Esgro recombinant mouse lif

WT and Txnip KO MEFs were derived from E13.5 mouse embryos and cultured in MEF medium (high glucose DMEM, 20% FBS, and 100 U penicillin and streptomycin). MEFs were seeded and delivered with competent sendai virus (SeV) that carry Yamanaka factors using CytoTune-iPS Sendai Reprogramming (Theromo) to reprogram MEFs into iPSCs, as previously reported [12 (link), 13 (link)]. iPSC colonies were picked and expanded for further iPSC characterization. PSC medium was composed of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with Fetal bovine serum (FBS) (15%, Gibco), Glutamax™ supplement (1%, Gibco), MEM Non-Essential Amino Acids (1%, Gibco), penicillin/streptomycin (1%, Gibco) and ESGRO recombinant mouse LIF (1000 U/ml, Millipore).

Mouse PSCs were mainly cultured in high glucose DMEM (Gibco, 11965092) supplemented with 15% FBS (Gibco), Glutamax™ supplement (1%, Gibco), β-mercaptoethanol (55 μM, Gibco), MEM non-essential amino acid (1%, Gibco), penicillin and streptomycin (1%, Gibco), and ESGRO recombinant mouse LIF (1000 U/ml, Millipore), as previously reported [14 (link)]. For low glucose conditions, high glucose DMEM (Gibco, 11965092) was mixed with no glucose DMEM (Gibco, 11966025) in 1:4 ratio, generating final glucose concentration of 5 mM. Spontaneous differentiation of PSCs was induced by LIF withdrawal from PSC medium and addition of retinoic acid (RA, final concentration 0.5 µM) in monolayer cultures. For embryonic body (EB) formation, PSCs were cultured in low-attachment dishes that contained PSC medium without LIF. Briefly, PSCs were trypsinized and cultivated as EBs by hanging drop method (500 cells/10 µl drop) in the absence of LIF for 2 days followed by 4-day suspension culture.