A fibroblast-derived, integration-free iPSC line generated using episomal plasmids was differentiated to definitive endoderm by 3 days of exposure to 100ng/mL Activin A. Generation of definitive endoderm was confirmed by detection of co-expression of FoxA2 and Sox17. This method provides the foundation for any protocol to generate endoderm-derived cell types.
• D'Amour, et al. "Efficient differentiation of human embryonic stem cells to definitive endoderm." Nat Biotechnol 23(12): 1534-1541 (2005).
H9 hESCs were differentiated to hepatocyte-like cells by generation of definitive endoderm followed by sequential exposure to FGF2/BMP4, HGF, and oncostatin M. Immature hepatocytes expressing AFP and albumin are shown. Cells generated with this protocol have been reported to possess many key hepatocyte functions in vitro, integrate into mouse liver parenchyma, and exhibit prolonged secretion of human albumin in vivo. These cells support the entire life cycle of hepatitis C virus in vitro and have recently been used to identify a key regulator of human hepatocyte development.
• Si-Tayeb et al. "Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells." Hepatology51(1): 297-305 (2010).
eratinocyte-derived iPSCs were first differentiated to definitive endoderm and then exposed to FGF4/Wnt3a to generate hindgut endoderm which undergoes morphogenesis to form free-floating spheroids. Following culture in pro-intestinal conditions organoids are generated that contain all major intestinal epithelial cell types enterocytes (villin), goblet cells (mucin), paneth cells (lysozyme), and enteroendocrine cells (chromogranin A). Epithelia are organized into crypt/villus-like structures, and contain proliferative progenitor domains. Enterocytes have typical brush border and microvilli and exhibit functional peptide transport system. This model is being used to study multiple aspects of human intestine development, homeostasis and disease.
• Spence et al. "Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro." Nature 470(7332): 105-109 (2011).