Objectives Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can generate any given cell type in the human body. study was to identify and validate small molecules that can induce DE differentiation and further enhance pancreatic progenitor differentiation. Therefore, we developed a large scale, high-content screen for testing a chemical library of 23,406 small molecules to identify compounds that induce FoxA2 in mouse embryonic stem cells (mESCs). Results Based on Byakangelicol IC50 our high-content screen algorithm, we selected 84 compounds that directed differentiation of mESCs towards the FoxA2 lineage. Strikingly, we identified ROCK inhibition (ROCKi) as a novel mechanism of endoderm induction in mESCs and hESCs. DE induced by the ROCK inhibitor Fasudil efficiently gives rise to PDX1+ pancreatic progenitors from hESCs. Conclusion Taken together, DE induction by ROCKi can simplify and improve current endoderm and pancreatic differentiation protocols towards a GMP-grade cell product for -cell replacement. JNKK1 to facilitate generation and upscaling of pancreatic -cells , . A major drawback of these protocols is the use of recombinant proteins and ligands that show variable activity and stability and are often exposed to animal products that might be contaminated with yet unidentified pathogens , . One strategy to overcome this problem and implement cheap and efficient GMP-grade ESC differentiation protocols is to replace biologics by small molecule compounds with stable and reproducible activity. During embryogenesis, different developmental pathways regulate definitive endoderm (DE) formation and patterning, including the Wnt, fibroblast growth factor (FGF), transforming growth factor (TGF- )/Nodal/ActivinA (AA), bone morphogenic protein (BMP), and AKT/PI3K , , , . Modulating the signaling transduction events and genes involved in these Byakangelicol IC50 pathways can help recapitulate the developmental processes from one week to another. Induction of heterogeneous DE populations can lead to a great inconsistency in establishing long-term differentiation protocols over 20C40 days towards one particular cell fate , . Small molecules can serve as tools to replace current proteins and induce the differentiation of ESCs. These molecules can effectively act on target proteins thereby modulating different signaling pathways . The major advantage of using small molecules is that they can be synthesized in high amounts and with higher purity and stored in a way that the substances have reproducible activity. High-throughput screens to monitor directed endodermal differentiation have been reported previously , . These screens introduce small molecules that modulate the TGF- pathway, replacing the use of AA in differentiation cocktails to induce endoderm; however, there is still a great need to identify novel Byakangelicol IC50 potent endoderm inducers that can effectively augment terminal pancreatic differentiation protocols , , , , . Towards this aim, we set-up a high-content screen in mESCs and tested 23,406 small molecules. We identified the Rho associated coiled like protein kinase (ROCK) inhibitor Fasudil as a small molecule that efficiently induces DE in both mESCs and hESCs. Moreover, when compared with the traditional Wnt3a and AA endoderm induction cocktail, ROCKi treated cells showed similar differentiation towards DE. We show that another analogue of Fasudil, RKI-1441, showed similar differentiation efficiencies of mESCs and hESCs towards DE indicating that ROCKi is sufficient to induce DE in culture. Furthermore, the ROCKi differentiates the PSCs towards anterior definitive endoderm (ADE), which gives rise to thymus, thyroid, lung, liver, and pancreas. We found that ROCKi does not induce extraembryonic visceral endoderm or mesoderm in the cell culture system. Additionally, ROCKi-induced DE from hESCs differentiated efficiently into pancreatic progenitors (PP), suggesting a supportive role of ROCKi in pancreatic differentiation. Altogether, we introduce a family of small molecule ROCKis and a novel mechanism that can robustly induce DE/ADE differentiation of PSCs in culture thereby replacing biologics in the differentiation medium. 2.?Methods and materials 2.1. Culture, maintenance, and differentiation of mouse and human embryonic stem cells In-house made (IDG) mESCs (FoxA2-Venus/Oct3/4-RFP) were thawed.