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

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.

Stem Cells Therapy in Diabetes Mellitus

“…Fibroblast growth factor (FGF) 10, retinoic acid and activin are used to induce the differentiation of hESCs into Pdx1 expressing cells [28][29][30]. Other markers used to identify definitive endoderm include SOX17, brachyury protein, FGF7, FoXa2, CXC-chemokine receptor (CXCr) 4 and Cerberus [31][32][33][34][35]. Definitive endoderm 1 and 2 (iDe1 and iDe2) have been shown to induce the construction of ultimate endoderm from mouse and human ESCs with about 70-80% efficiency, which is much higher than the differentiation induced by activin or nodal [36,37].…”

Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

“…Finally, cell density is only one of the various factors involved in the neural differentiation of human ESCs. It has been combined with other factors, such as growth factors[7], colony and aggregate size[10], expansion time[22], seeding protocol[23] and matrix concentration[24], to play a crucial role in the differentiation of various stem cells, including the neural differentiation of human ESCs. Thus, it also remains to be elucidated how cell density, particularly LCD, cooperates with other factors involved in NE induction to generate optimal outcomes.…”

“…For human ESCs, high density culturing generates central nervous system (CNS)-neuronal derivatives, while lower density conditions favor peripheral nervous system (PNS) development [ 6 ]. Nevertheless, high cell seeding densities is required for the final differentiation of pancreatic amylase-positive cells from human ESCs [ 7 ]. High density cultures also favor pancreatic progenitor commitment and an increased formation of pancreatic endocrine cell populations [ 5 ].…”

Patients' Induced Pluripotent Stem Cells to Model Drug Induced Adverse Events: A Role in Predicting Thiopurine Induced Pancreatitis?

“…Step III, differentiation of pancreatic exocrine cells by exposure to fibroblast growth factor 7 (FGF7), glucagon-like peptide 1 (GLP-1) and nicotinamide (NA) in combination [63]. In [63]; these cells could be tested as an in vitro model of drug induced pancreatitis and in particular TIP.…”