In vitro differentiation of exocrine pancreatic cells from mouse embryonic stem cells

AIM: To explore the effects of sodium butyrate, activin A and dexamthasone on inducing mouse embryonic stem (ES) cells to differentiate into exocrine pancreatic cells in vitro. METHODS: E14 mouse ES cells were cultured in suspension to form embryonic bodies (EBs). The EBs were cultured with differentiating medium containing different concentrations of sodium butyrate, and the spontaneously differentiated ES cells were used as control. Exocrine pancreatic genes such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR at different time points to determine the optimal concentration and exposure time of sodium butyrate. Furthermore, activin A or dexamthasone was also used to explore the effects on exocrine differentiation. After that, the combination of sodium butyrate, activin A and dexamthasone was used to promote the differentiation of exocrine pancreatic cells from ES cells. During the differentiation course, the gene expressions of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR. Morphological changes were investigated by phase contrast microscopy. Amylase expression was examined by immunofluorescence staining. RESULTS: Exocrine pancreatic gene expressions such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected in spontaneously differentiated EBs. A relatively lower concentration of sodium butyrate with a shorter exposure time significantly promoted those above gene expressions as compared to that of spontaneously differentiated EBs. Activin A and dexamethasone induced upregulation of exocrine gene expression. The combination of activin A, sodium butyrate and dexamethasone significantly enhanced the mRNA levels of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase. Under the treatment of activin A, sodium butyrate and dexamethasone, differentiated cells were polygonal in shape with large, round, and center-situated nuclei. According to the observation of immunofluorescence staining, amylase was positive expressed at the final stage. CONCLUSION: These data indicate that exocrine pancreatic differentiation of ES cells is induced by sodium butyrate, activin A and dexamethasone. The combination of pancreatic inducing factors improves the differentiating efficiency.