Abstract
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.
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Bayha E, Jørgensen MC, Serup P, Grapin-Botton A (2009) Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis. PLoS One 10:e5845
Cai J, Yu C, Liu Y, Chen S, Guo Y, Yong J, Lu W, Ding M, Deng H (2010) Generation of homogenous PDX1(+) pancreatic progenitors from human ES cell-derived endoderm cells. J Mol Cell Biol 2:50–60
Chen S, Borowial M, Fox JL, Maehr R, Osafune K, Davidow L, Lam K, Peng LF, Schreiber SL, Rubin LL, Melton D (2009) A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol 5:258–265
Chen YF, Tseng CY, Wang HW, Kuo HC, Yang VW, Lee OK (2012) Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. Hepatology 55:1193–1203
Cleveland MH, Sawyer JM, Afelik S, Jensen J, Leach SD (2012) Exocrine ontogenies: on the development of pancreatic acinar, ductal and centroacinar cells. Semin Cell Dev Biol 23:711–719
Coffinier C, Thépot D, Babinet C, Yaniv M, Barra J (1999) Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation. Development 126:4785–4794
D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE (2005) Efficient differentiation of human embryonic stem cells to definitve endoderm. Nat Biotechnol 23:1534–1541
D’Amour KA, Bang AG, Eliazer S, Kelly OG, Aqulnick AD, Smart NG, Moorman MA, Kroon E, Carpenter MK, Baetge EE (2006) Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24:1392–1401
Elqhazi L, Cras-Méneur C, Czernichow P, Scharfmann R (2002) Role for FGFR2IIIb-mediated signaling in controlling pancreatic endocrine progenitor cell differentiation. Proc Natl Acad Sci U S A 99:3884–3889
Fu S, Fei Q, Jiang H, Chuai S, Shi S, Xiong W, Jiang L, Lu C, Atadja P, Li E, Shou J (2011) Involvement of histone acetylation of Sox17 and Foxa2 promoters during mouse definitive endoderm differentiation revealed by microRNA profiling. PLoS One 6:e27965
Hong SG, Dunbar CE, Winkler T (2012) Assessing the risks of genotoxycity in the therapeutic development of induced pluripotent stem cells. Mol Ther 21:272–281
Micallef SJ, Janes ME, Knezevic K, Davis RP, Elefantry AG, Stanley EG (2005) Retinoic acid induced Pdx1-positive endoderm in differentiating mouse embryonic stem cells. Diabetes 54:301–305
Miralles F, Czernichow P, Ozaki K, Itoh N, Scharfmann R (1999) Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas. Proc Natl Acad Sci U S A 96:6267–6272
Mfopou JK, Chen B, Mateizel I, Sermon K, Bouwens L (2010) Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells. Gastroenterology 138:2233–2245
Okita K, Nagata N, Yamanaka S (2011) Immunogenicity of induced pluripotent stem cells. Circ Res 109:720–721
Poll AV, Pierreux CE, Lokmane L, Haumaitre C, Achouri Y, Jacquemin P, Rousseau GG, Careqhini S, Lemaiqre FP (2006) A vHNF1/TCF2-HNF6 cascade regulates the transcription factor network that controls generation of pancreatic precursor cells. Diabetes 55:61–69
Rovira M, Delaspre F, Massumi M, Serra SA, Valverde MA, Lloreta J, Dufresne M, Payré B, Konieczy SF, Savatier P, Real FX, Skoudy A (2008) Murine embryonic stem cell-derived pancreatic acinar cells recapitulated features of early pancreatic differentiation. Gastroenterology 135:1301–1310
Shirasawa S, Yoshie S, Yokoyama T, Tomotsune D, Yue F, Sasaki K (2011a) A novel stepwise differentiation of functional pancreatic exocrine cells from embryonic stem cells. Stem Cells Dev 20:1071–1078
Shirasawa S, Yoshie S, Yue F, Ichikawa H, Yokoyama T, Nagai M, Tomotsune D, Hirayama M, Sasaki K (2011b) Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells. Biochem Biophys Res Commun 410:608–613
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 30:861–872
Thatava T, Nelson TJ, Edukulla R, Sakuma T, Ohmine S, Tonne JM, Yamada S, Kudva Y, Terzic A, Ikeda Y (2011) Indolactam V/GLP-1-mediated differentiation of human iPS cells into glucose-responsive insulin-secreting progeny. Gene Ther 18:283–293
Ye F, Duvillié B, Scharfmann R (2005) Fibroblast growth factors 7 and 10 are expressed in the embryonic pancreatic mesenchyme and promote the proliferation of embryonic pancreatic epitherial cells. Diabetologia 48:277–281
Zhang D, Jiang W, Liu M, Sui X, Yin X, Chen S, Shi Y, Deng H (2009) Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res 19:429–438
Zhao T, Zhang ZN, Rong Z, Xu Y (2011) Immunogenicity of induced pluripotent stem cells. Nature 474:212–215
Acknowledgment
The authors thank Mr. Kayo Suzuki and Dr. Kiyokazu Kametani (Research Center for Instrumental Analysis, Shinshu University) for their outstanding technical assistance.
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Takizawa-Shirasawa, S., Yoshie, S., Yue, F. et al. FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells. Cell Tissue Res 354, 751–759 (2013). https://doi.org/10.1007/s00441-013-1695-6
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DOI: https://doi.org/10.1007/s00441-013-1695-6