Rho kinase inhibitor

iPSCs were single-cell dissociated using Accutase and plated onto Matrigel-coated plates at a density of 300,000 cells/cm² using mTeSR⁺ and 10 µM Rho kinase Inhibitor (Stem Cell Tech). The following day, cells were directed into Definitive Endoderm (DE) using Phase I medium, which was composed of base medium MCDB 131 (Fisher Sci) supplemented with 100 ng/ml Activin A (R&D), 2 µM CHIR99021 (Stemgent), and 10 µM Rho kinase Inhibitor (Stem Cell Tech.) for 1 day. For the next two days, the same base medium was used, but supplemented instead with 100 ng/mL Activin A and 5 ng/mL FGF2 (Peprotech). Following this phase, cells were directed to form Posterior Foregut (PFG) using Phase II medium, which was composed of the same base medium as Phase I but supplemented with 50 ng/mL FGF10 (Peprotech), 0.25 µM CHIR99021 and 50 ng/ml Noggin (Peprotech), for 2 days. To reach a Pancreatic Progenitor (PP) stage, cells were fed with Phase III medium, which was composed of DMEM supplemented with 50 ng/mL Noggin, 50 ng/mL FGF10, 2 µM Retinoic Acid (Sigma), and 0.25 µM SANT1 (Sigma), for four days. More details about base medium formulation are described at Supplementary Table 1. This protocol was based on a published protocol to differentiate iPSCs into Pancreatic Progenitors (Memon et al., 2018 (link)).