Ac93253

We conducted detailed studies of chemical toxicity on hiPSCs, hMSCs, and their differentiated progenies at various stages of differentiation (Figure S1). Cells were treated with different chemicals in 96-well plates and cell viability was measured with the CCK-8 assay (Sigma-Aldrich, Cat. No. 96992). For all chemical toxicity assays, 6,000 hMSCs in 100 μL medium were seeded per well in a 96-well plate, and treated for 48 hours at 37°C/5% CO₂ before cell viability readout on a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA). A total of 14 different concentrations ranged from 0.0029 to 223 μM were used for each chemical in triplicates. The four chemicals, AC-93253, idarubicin, NSC 95397, and cantharidin, were purchased from Sigma, and dissolved in 100% DMSO. For chemical toxicity test on hMSCs, the final DMSO concentration in the media was 0.5%. For hiPSCs and hepatic differentiation Phase I cells, the final DMSO concentration was 0.1%. For hepatic differentiation Phase II and Phase III chemical toxicity tests, the final DMSO concentration for NSC 95397 was 0.3%, while 0.1% for the other 3 chemicals. Each chemical toxicity tests were repeated at least three times. The cell viabilities were plotted versus concentrations for each chemical. GraphPad Prism 6 was used to fit the data to a 3-parameter dose-inhibition curve to obtain the IC₅₀ values.