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GSK3 inhibitor-BIO regulates proliferation of immortalized pancreatic mesenchymal stem cells (iPMSCs).

Cao H, Chu Y, Lv X, Qiu P, Liu C, Zhang H, Li D, Peng S, Dou Z, Hua J - PLoS ONE (2012)

Bottom Line: The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs).However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present.However, we did not find the related roles of BIO on β cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering and Technology, Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A & F University, Yangling, Shaanxi, People's Republic of China.

ABSTRACT

Background: The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs). However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present.

Results: To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effect of BIO on iPMSCs. We found that the inactivation of GSK3 by BIO can robustly stimulate iPMSCs proliferation and mass formation as shown by QRT-PCR, western blotting, 5-Bromo-2-deoxyuridine (BrdU) immunostaining assay and tunel assay. However, we did not find the related roles of BIO on β cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis.

Conclusions: These results suggest that BIO plays a key role in the regulation of cell mass proliferation and maintenance of the undifferentiated state of iPMSCs.

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Potential differentiation of islet cells.A, The induced clusters treated with BIO or not were transferred into plates and stained positive for PDX1, C-peptide and insulin analysed by immunofluorescent staining; B, The specific markers of pancreatic islets including PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin expressed in the iPMSCs treated with BIO or not by QRT-PCR; C, Glucose-stimulated insulin release and insulin content analysis. Bar = 20 µm, **P<0.01.
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pone-0031502-g006: Potential differentiation of islet cells.A, The induced clusters treated with BIO or not were transferred into plates and stained positive for PDX1, C-peptide and insulin analysed by immunofluorescent staining; B, The specific markers of pancreatic islets including PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin expressed in the iPMSCs treated with BIO or not by QRT-PCR; C, Glucose-stimulated insulin release and insulin content analysis. Bar = 20 µm, **P<0.01.

Mentions: BIO stimulated iPMSCs to form smaller, denser and more compact colonies (Figure 1). Further, we investigated the islet potential differentiation of iPMSCs in the absence or presence of BIO by inducing the cells with a two-step protocol [7]. On the 14th day, induced pancreatic islet-like clusters were formed. Islet-like formation derived from iPMSCs in containing BIO induced medium were denser (Figure 6). After two weeks, the induced clusters were transferred into plates and stained positive for PDX1, C-peptide and Insulin, as analysed by immunofluorescent staining (Figure 6A). These results provide evidences that the cells in the induction medium with or without BIO have the potential to differentiate into islet cell clusters and endocrine cell types in this protocol [21], and the number of islet cell clusters derived from iPMSCs were slightly increased in BIO induced medium. At the mRNA level, we detected endocrine pancreatic islet markers including PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin expressed in iPMSCs treated with or without BIO, however, BIO treatment did not alter the expression levels of PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin analysed by QRT-PCR (Figure 6B).


GSK3 inhibitor-BIO regulates proliferation of immortalized pancreatic mesenchymal stem cells (iPMSCs).

Cao H, Chu Y, Lv X, Qiu P, Liu C, Zhang H, Li D, Peng S, Dou Z, Hua J - PLoS ONE (2012)

Potential differentiation of islet cells.A, The induced clusters treated with BIO or not were transferred into plates and stained positive for PDX1, C-peptide and insulin analysed by immunofluorescent staining; B, The specific markers of pancreatic islets including PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin expressed in the iPMSCs treated with BIO or not by QRT-PCR; C, Glucose-stimulated insulin release and insulin content analysis. Bar = 20 µm, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285662&req=5

pone-0031502-g006: Potential differentiation of islet cells.A, The induced clusters treated with BIO or not were transferred into plates and stained positive for PDX1, C-peptide and insulin analysed by immunofluorescent staining; B, The specific markers of pancreatic islets including PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin expressed in the iPMSCs treated with BIO or not by QRT-PCR; C, Glucose-stimulated insulin release and insulin content analysis. Bar = 20 µm, **P<0.01.
Mentions: BIO stimulated iPMSCs to form smaller, denser and more compact colonies (Figure 1). Further, we investigated the islet potential differentiation of iPMSCs in the absence or presence of BIO by inducing the cells with a two-step protocol [7]. On the 14th day, induced pancreatic islet-like clusters were formed. Islet-like formation derived from iPMSCs in containing BIO induced medium were denser (Figure 6). After two weeks, the induced clusters were transferred into plates and stained positive for PDX1, C-peptide and Insulin, as analysed by immunofluorescent staining (Figure 6A). These results provide evidences that the cells in the induction medium with or without BIO have the potential to differentiate into islet cell clusters and endocrine cell types in this protocol [21], and the number of islet cell clusters derived from iPMSCs were slightly increased in BIO induced medium. At the mRNA level, we detected endocrine pancreatic islet markers including PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin expressed in iPMSCs treated with or without BIO, however, BIO treatment did not alter the expression levels of PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin analysed by QRT-PCR (Figure 6B).

Bottom Line: The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs).However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present.However, we did not find the related roles of BIO on β cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering and Technology, Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A & F University, Yangling, Shaanxi, People's Republic of China.

ABSTRACT

Background: The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs). However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present.

Results: To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effect of BIO on iPMSCs. We found that the inactivation of GSK3 by BIO can robustly stimulate iPMSCs proliferation and mass formation as shown by QRT-PCR, western blotting, 5-Bromo-2-deoxyuridine (BrdU) immunostaining assay and tunel assay. However, we did not find the related roles of BIO on β cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis.

Conclusions: These results suggest that BIO plays a key role in the regulation of cell mass proliferation and maintenance of the undifferentiated state of iPMSCs.

Show MeSH
Related in: MedlinePlus