FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells

• Published in: Cell and tissue research 2013-12, Vol.354 (3), p.751-759
• Main Authors: Takizawa-Shirasawa, Sakiko, Yoshie, Susumu, Yue, Fengming, Mogi, Akimi, Yokoyama, Tadayuki, Tomotsune, Daihachiro, Sasaki, Katsunori
• Format: Article
• Language: English
• Subjects: Activins - pharmacology; Amylases; Amylases - biosynthesis; Biomedical and Life Sciences; Biomedicine; carboxypeptidase A; Cell Communication - drug effects; Cell Communication - physiology; Cell Count; Cell Culture Techniques - methods; Cell Differentiation - drug effects; Cell Differentiation - physiology; Cells, Cultured; Cellular biology; chymotrypsinogen; Cytological Techniques; Embryonic stem cells; Embryonic Stem Cells - cytology; Embryonic Stem Cells - drug effects; Embryonic Stem Cells - metabolism; Fibroblast Growth Factor 7 - genetics; Fibroblast Growth Factor 7 - metabolism; Fibroblast Growth Factor 7 - pharmacology; Fibroblast growth factors; fibroblasts; Gene expression; glucagon-like peptide 1; Glucagon-Like Peptide 1 - pharmacology; Human Genetics; Humans; immunohistochemistry; Molecular Medicine; Niacinamide; Niacinamide - pharmacology; nicotinamide; Pancreas; Pancreas, Exocrine - cytology; Pancreas, Exocrine - drug effects; Pancreas, Exocrine - enzymology; Pancreas, Exocrine - metabolism; Proteins; Proteomics; Regular Article; retinoic acid; Stem cells; Tretinoin - pharmacology
• ISSN: 0302-766X; 1432-0878
• DOI: 10.1007/s00441-013-1695-6

The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.