Comparable Generation of Activin-Induced Definitive Endoderm via Additive Wnt or BMP Signaling in Absence of Serum

•High dose of WNT3A cooperates with Activin to induce DE without serum•GSK-3 inhibitors, CHIR99021 and BIO, work with Activin to induce DE without serum•Wnt and BMP signaling can induce DE with comparable efficiencies without serum•WNT3A is not as effective as BMP4, CHIR99021, or BIO in suppressing pluripotency

SummaryThere is considerable interest in differentiating human pluripotent stem cells (hPSCs) into definitive endoderm (DE) and pancreatic cells for in vitro disease modeling and cell replacement therapy. Numerous protocols use fetal bovine serum, which contains poorly defined factors to induce DE formation. Here, we compared Wnt and BMP in their ability to cooperate with Activin signaling to promote DE formation in a chemically defined medium. Varying concentrations of WNT3A, glycogen synthase kinase (GSK)-3 inhibitors CHIR99021 and 6-bromoindirubin-3′-oxime (BIO), and BMP4 could independently co-operate with Activin to effectively induce DE formation even in the absence of serum. Overall, CHIR99021 is favored due to its cost effectiveness. Surprisingly, WNT3A was ineffective in suppressing E-CADHERIN/CDH1 and pluripotency factor gene expression unlike GSK-3 inhibitors or BMP4. Our findings indicate that both Wnt and BMP effectively synergize with Activin signaling to generate DE from hPSCs, although WNT3A requires additional factors to suppress the pluripotency program inherent in hPSCs.

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