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

Ren M, Yan L, Shang CZ, Cao J, Li FP, Li JY, Cheng H, Min J - Acta Pharmacol. Sin. (2009)

Bottom Line: The effects of activinA and dexamethasone (Dex) on exocrine differentiation were also explored.Further combination with Dex and other pancreatic-inducing factors, such as activinA, significantly enhanced the mRNA and protein levels of exocrine pancreatic markers.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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Endocrinology and Hepatobiliary Surgery, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, China.

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.

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Related in: MedlinePlus

Expression of exocrine markers in EBs incubated with activinA, sodium butyrate and Dex. EBs grown in suspension were cultured in 25 ng/mL activinA for 48 h, followed by 1 mmol/L sodium butyrate treatment for 5 days. At the final stage of differentiation, 10−7 mol/L Dex was added for another 7 days. RNA from both spontaneously differentiated and treated cells was isolated and RT-PCR was performed to measure the mRNA levels of exocrine genes. The ratios of exocrine markers to β-actin were calculated. Values are given as means±SD. (A) A representative PCR result is shown. (B) A histogram plot representing densitometric analysis, corresponding to the means±SD of three independent experiments.
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fig5: Expression of exocrine markers in EBs incubated with activinA, sodium butyrate and Dex. EBs grown in suspension were cultured in 25 ng/mL activinA for 48 h, followed by 1 mmol/L sodium butyrate treatment for 5 days. At the final stage of differentiation, 10−7 mol/L Dex was added for another 7 days. RNA from both spontaneously differentiated and treated cells was isolated and RT-PCR was performed to measure the mRNA levels of exocrine genes. The ratios of exocrine markers to β-actin were calculated. Values are given as means±SD. (A) A representative PCR result is shown. (B) A histogram plot representing densitometric analysis, corresponding to the means±SD of three independent experiments.

Mentions: Because a single inducing agent cannot achieve high differentiation efficiency, a combination of differentiation agents was needed to enhance exocrine pancreatic differentiation. To induce final differentiation, activinA was used for the first 2 d, followed by the addition of sodium butyrate for 5 d. Finally, following activinA and sodium butyrate treatment, the differentiated cells were transferred into Dex-containing medium for another 7 d. Compared with activinA treatment alone, the combination of sodium butyrate and activinA resulted in higher levels of exocrine gene expression (Figure 5). Final treatment with Dex induced more marked amylase, elastase, and chymotrypsinogen gene expression compared with treatment with activinA and sodium butyrate alone. Meanwhile, exocrine gene expression in spontaneously differentiated cells clearly decreased over the same 14-d culture duration (Figure 5).


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

Ren M, Yan L, Shang CZ, Cao J, Li FP, Li JY, Cheng H, Min J - Acta Pharmacol. Sin. (2009)

Expression of exocrine markers in EBs incubated with activinA, sodium butyrate and Dex. EBs grown in suspension were cultured in 25 ng/mL activinA for 48 h, followed by 1 mmol/L sodium butyrate treatment for 5 days. At the final stage of differentiation, 10−7 mol/L Dex was added for another 7 days. RNA from both spontaneously differentiated and treated cells was isolated and RT-PCR was performed to measure the mRNA levels of exocrine genes. The ratios of exocrine markers to β-actin were calculated. Values are given as means±SD. (A) A representative PCR result is shown. (B) A histogram plot representing densitometric analysis, corresponding to the means±SD of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007190&req=5

fig5: Expression of exocrine markers in EBs incubated with activinA, sodium butyrate and Dex. EBs grown in suspension were cultured in 25 ng/mL activinA for 48 h, followed by 1 mmol/L sodium butyrate treatment for 5 days. At the final stage of differentiation, 10−7 mol/L Dex was added for another 7 days. RNA from both spontaneously differentiated and treated cells was isolated and RT-PCR was performed to measure the mRNA levels of exocrine genes. The ratios of exocrine markers to β-actin were calculated. Values are given as means±SD. (A) A representative PCR result is shown. (B) A histogram plot representing densitometric analysis, corresponding to the means±SD of three independent experiments.
Mentions: Because a single inducing agent cannot achieve high differentiation efficiency, a combination of differentiation agents was needed to enhance exocrine pancreatic differentiation. To induce final differentiation, activinA was used for the first 2 d, followed by the addition of sodium butyrate for 5 d. Finally, following activinA and sodium butyrate treatment, the differentiated cells were transferred into Dex-containing medium for another 7 d. Compared with activinA treatment alone, the combination of sodium butyrate and activinA resulted in higher levels of exocrine gene expression (Figure 5). Final treatment with Dex induced more marked amylase, elastase, and chymotrypsinogen gene expression compared with treatment with activinA and sodium butyrate alone. Meanwhile, exocrine gene expression in spontaneously differentiated cells clearly decreased over the same 14-d culture duration (Figure 5).

Bottom Line: The effects of activinA and dexamethasone (Dex) on exocrine differentiation were also explored.Further combination with Dex and other pancreatic-inducing factors, such as activinA, significantly enhanced the mRNA and protein levels of exocrine pancreatic markers.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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Endocrinology and Hepatobiliary Surgery, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, China.

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.

Show MeSH
Related in: MedlinePlus