Sodium butyrate and dexamethasone promote exocrine pancreatic gene expression in mouse embryonic stem cells

Abstract

Aim: The feasibility of inducing endocrine pancreatic differentiation of embryonic stem (ES) cells has been well documented. However, whether ES cells possess the potential for exocrine pancreatic differentiation requires further exploration. Here, we investigated whether sodium butyrate and glucocorticoids were conducive to the exocrine pancreatic differentiation of ES cells. Methods: E14 mouse ES cells were cultured in suspension to form embryoid bodies (EBs). These EBs were cultured in differentiating medium containing varying concentrations of sodium butyrate. The effects of activinA and dexamethasone (Dex) on exocrine differentiation were also explored. Finally, the combination of sodium butyrate, activinA, and Dex was used to promote the differentiation of exocrine pancreatic cells. Specific exocrine pancreatic gene expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and amylase expression was examined by immunofluorescence staining. Flow cytometry analysis was also performed to determine the percentage of amylase-positive cells after the treatment with activinA, sodium butyrate, and Dex. Results: Exposure of ES cells to 1 mmol/L sodium butyrate for 5 days promoted exocrine pancreatic gene expression. Further combination with Dex and other pancreatic-inducing factors, such as activinA, significantly enhanced the mRNA and protein levels of exocrine pancreatic markers. Additionally, flow cytometry revealed that approximately 17% of the final differentiated cells were amylase-positive. Conclusion: These data indicate that the exocrine pancreatic differentiation of ES cells can be induced by activinA, sodium butyrate, and Dex, providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.

Directed Pancreatic Acinar Differentiation of Mouse Embryonic Stem Cells via Embryonic Signalling Molecules and Exocrine Transcription Factors

“…Therefore, to sustain exocrine differentiation in ESC cultures, cells were simultaneously treated with both factors as it is unlikely that with one single differentiating agent a robust exocrine differentiation would have been achieved [30]. In agreement with the results of Ren et al [15], dexamethasone was crucial for an optimal induction of digestive enzyme expression but only when added in combination with the other factors. In this respect, co-treatment with follistatin and FGF7 selectively increased the expression of these markers but to a lesser extent (data not shown).…”

“…An important aspect that has not been previously studied refers to the generally accepted notion that prolonged exposure to glucocorticoids results in the reprogramming of acinar cells into hepatic-like cells [15], [53]. In our study, the drastic increase in digestive enzymes was not accompanied by a strong rise of hepatic markers (Fig.…”

“…In this respect, co-treatment with follistatin and FGF7 selectively increased the expression of these markers but to a lesser extent (data not shown). However, the previous combination of factors (Activin A+sodium butyrate+dexamethasone) was somewhat quite inefficient resulting in nearly two-fold increase in the induction of digestive enzymes as compared with control cultures [15]. By contrast, in our experiments there was a substantial increase in the efficiency of induction of digestive enzymes (a factor of 10 3 –10 4 times as an average estimation) with respect to cultures only treated with 1% SR, which by itself is permissive on ESC acinar differentiation [49].…”

“…In this sense, one important aspect missing in many pancreatic differentiation protocols is to assess the extent of selectivity in cell lineage induction. In this regard, other studies have reported the expression of acinar markers from ESC by manipulating several developmental pathways already established for endocrine differentiation or without examining their role on endocrine gene expression [12], [13], [14], [15]. Therefore, progress in the knowledge of how acinar cells are formed during embryogenesis is essential for the improvement of strategies assessing ESC exocrine differentiation.…”

“…When this protocol was coupled to high expression of Ptf1a and Rbpjl (designed Ptf1a High and Rbpjl High ), an important rise in the expression of digestive enzymes was observed and cells became responsive to secretagogues. We believe that this new protocol is improved to others in that: i) it favours the generation of exocrine progenitors over the production of the endocrine lineage, ii) it leads to a more mature pattern of the regulatory digestive enzyme expression modules than other reported protocols [11], and iii) generates functional cells in a shorter time [11], [13], [14], [15]. Therefore, this approach might be instrumental for a gain of knowledge of the developmental acinar program and the establishment of in vitro models for the research on pancreatic exocrine disease.…”