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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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β-catenin and BrdU incorporation assay.A, The mitiosis index with BIO was significantly higher than without BIO medium; B, The percentage of positive cells treated with BIO or not from A; C, BIO treatment caused nuclear accumulation of β-catenin, which was not observed in control cells; D, Cells were assayed for β-catenin-mediated transcriptional activation by using a reporter containing either functional (TOP-Flash) or mutated (FOP-Flash) Tcf binding sites. Cells stimulated with BIO showed rise in the levels of β-catenin activity, as measured by luciferase activity. Treatment with BIO resulted in the higher fold change (almost 4 fold) when TOP-Flash activity was normalized against FOP-Flash activity. There was no significant difference in activities in the presence of the mutated FOP-Flash reporter; C, Bar = 20 µm, *P<0.05; E, Western blotting showed that p-β-catenin (S33/37/T41) was decreased in BIO treated.
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pone-0031502-g003: β-catenin and BrdU incorporation assay.A, The mitiosis index with BIO was significantly higher than without BIO medium; B, The percentage of positive cells treated with BIO or not from A; C, BIO treatment caused nuclear accumulation of β-catenin, which was not observed in control cells; D, Cells were assayed for β-catenin-mediated transcriptional activation by using a reporter containing either functional (TOP-Flash) or mutated (FOP-Flash) Tcf binding sites. Cells stimulated with BIO showed rise in the levels of β-catenin activity, as measured by luciferase activity. Treatment with BIO resulted in the higher fold change (almost 4 fold) when TOP-Flash activity was normalized against FOP-Flash activity. There was no significant difference in activities in the presence of the mutated FOP-Flash reporter; C, Bar = 20 µm, *P<0.05; E, Western blotting showed that p-β-catenin (S33/37/T41) was decreased in BIO treated.

Mentions: BrdU incorporation assay demonstrated that the mitosis index in the presence of BIO was significantly higher than without BIO (Figure 3A, P<0.05). Control cells had a lower rate of BrdU incorporation (31.7±1.3%). In contrast, stimulation of iPMSCs with 1 µM BIO increased the BrdU incorporation rate to 52.7±1.9% (Figure 3B). These results demonstrated that BIO enhanced the proliferation of iPMSCs.


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)

β-catenin and BrdU incorporation assay.A, The mitiosis index with BIO was significantly higher than without BIO medium; B, The percentage of positive cells treated with BIO or not from A; C, BIO treatment caused nuclear accumulation of β-catenin, which was not observed in control cells; D, Cells were assayed for β-catenin-mediated transcriptional activation by using a reporter containing either functional (TOP-Flash) or mutated (FOP-Flash) Tcf binding sites. Cells stimulated with BIO showed rise in the levels of β-catenin activity, as measured by luciferase activity. Treatment with BIO resulted in the higher fold change (almost 4 fold) when TOP-Flash activity was normalized against FOP-Flash activity. There was no significant difference in activities in the presence of the mutated FOP-Flash reporter; C, Bar = 20 µm, *P<0.05; E, Western blotting showed that p-β-catenin (S33/37/T41) was decreased in BIO treated.
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Related In: Results  -  Collection

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

pone-0031502-g003: β-catenin and BrdU incorporation assay.A, The mitiosis index with BIO was significantly higher than without BIO medium; B, The percentage of positive cells treated with BIO or not from A; C, BIO treatment caused nuclear accumulation of β-catenin, which was not observed in control cells; D, Cells were assayed for β-catenin-mediated transcriptional activation by using a reporter containing either functional (TOP-Flash) or mutated (FOP-Flash) Tcf binding sites. Cells stimulated with BIO showed rise in the levels of β-catenin activity, as measured by luciferase activity. Treatment with BIO resulted in the higher fold change (almost 4 fold) when TOP-Flash activity was normalized against FOP-Flash activity. There was no significant difference in activities in the presence of the mutated FOP-Flash reporter; C, Bar = 20 µm, *P<0.05; E, Western blotting showed that p-β-catenin (S33/37/T41) was decreased in BIO treated.
Mentions: BrdU incorporation assay demonstrated that the mitosis index in the presence of BIO was significantly higher than without BIO (Figure 3A, P<0.05). Control cells had a lower rate of BrdU incorporation (31.7±1.3%). In contrast, stimulation of iPMSCs with 1 µM BIO increased the BrdU incorporation rate to 52.7±1.9% (Figure 3B). These results demonstrated that BIO enhanced the proliferation of iPMSCs.

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