In vitro activity: ID-8 reversibly maintains the long-term culture of ESCs by sustaining Nanog gene expression through Sox2-Oct3/4 activation. ID-8 enhances Wnt-mediated hESC survival and proliferation via inhibition of DYRKs. The mechanism study indicates that ID-8 maintains the undifferentiated state in the presence of Wnt by modulating Wnt/β-catenin signaling and enhancing the CBP/β-catenin association in hESCs.

Cell Assay: Briefly, the hESCs in feeder-free culture are dissociated completely with 0.05% trypsin-EDTA and seeded at 104 cells per well in Matrigel-coated 6-well culture plates and cultured in MEF-CM. Various concentrations of Wnt3, IQ-1, ID-8, and/or ICG-001 are supplemented into the culture media at the onset of seeding and then continuously until the end of culturing. For all assays, the cell and colony morphology are examined under a microscope, and the replating efficiency is examined by counting the number of colonies after 7 days of culture.