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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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Morphology of iPMSCs in the absence or presence of BIO.A-Control, The typical spindle fibroblast-like morphology of iPMSCs; A-BIO, The morphology of iPMSCs treated with BIO; B, iPMSCs stained with Giemsa in the absence or presence of BIO; C, The number of cells cultured in the absence or presence of BIO (0.1, 0.5, 1.0, 1.5, 2.0 µM); D, The number of iPMSCs colonies; E, Expression of PDX1, PCNA, Ngn3, TERT and C-Myc were analysed at mRNA level in the absence and presence of 1.0 and 1.5 µM BIO; F, The growth curves of iPMSCs cultured in the absence and presence of BIO (1 µM). Bar = 20 µm. * P<0.05, **, P<0.01.
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pone-0031502-g001: Morphology of iPMSCs in the absence or presence of BIO.A-Control, The typical spindle fibroblast-like morphology of iPMSCs; A-BIO, The morphology of iPMSCs treated with BIO; B, iPMSCs stained with Giemsa in the absence or presence of BIO; C, The number of cells cultured in the absence or presence of BIO (0.1, 0.5, 1.0, 1.5, 2.0 µM); D, The number of iPMSCs colonies; E, Expression of PDX1, PCNA, Ngn3, TERT and C-Myc were analysed at mRNA level in the absence and presence of 1.0 and 1.5 µM BIO; F, The growth curves of iPMSCs cultured in the absence and presence of BIO (1 µM). Bar = 20 µm. * P<0.05, **, P<0.01.

Mentions: iPMSCs presented as the typical triangles and long spindle cell morphology of mesenchymal-like cells in the presence and absence of BIO (Figure 1A). Different concentrations of BIO (0, 0.1, 0.5, 1.0, 1.5, 2.0 µM) were added to the culture medium to evaluate their effects based on the morphology of iPMSCs colonies stained with Giemsa. Cell colonies were significantly denser and bigger in BIO than without BIO (Figure 1B). iPMSCs cultured in the presence of BIO (0.1, 0.5, 1.0, 1.5, 2.0 µM) for 3 and 7 days in vitro proliferated faster than that in the absence of BIO (Figure 1C). The number of cells in the presence of BIO (1.0, 1.5 µM) was elevated 1.2–2 fold compared with that in the absence of BIO. But when treated with 2.0 µM BIO, the number of iPMSCs decreased. In addition, the number of colonies was significantly increased (about 2 fold, P<0.01) in the presence of BIO (Figure 1D). The results showed that BIO could promote the proliferation of iPMSCs, and we concluded that the optimal concentrations were 1.0 and 1.5 µM. Then we detected specific pancreatic stem cell markers at the mRNA level. Expression of PDX1, Ngn3, PCNA and C-Myc showed an upward trend in iPMSCs treated with 1.0 and 1.5 µM BIO (Figure 1E) as compared with the control group. And the expression of C-Myc was clearly increased in the presence of 1.0 µM BIO compared with 1.5 µM and control. Thus, we used 1.0 µM BIO in the following experiments.


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)

Morphology of iPMSCs in the absence or presence of BIO.A-Control, The typical spindle fibroblast-like morphology of iPMSCs; A-BIO, The morphology of iPMSCs treated with BIO; B, iPMSCs stained with Giemsa in the absence or presence of BIO; C, The number of cells cultured in the absence or presence of BIO (0.1, 0.5, 1.0, 1.5, 2.0 µM); D, The number of iPMSCs colonies; E, Expression of PDX1, PCNA, Ngn3, TERT and C-Myc were analysed at mRNA level in the absence and presence of 1.0 and 1.5 µM BIO; F, The growth curves of iPMSCs cultured in the absence and presence of BIO (1 µM). Bar = 20 µm. * P<0.05, **, P<0.01.
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Related In: Results  -  Collection

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pone-0031502-g001: Morphology of iPMSCs in the absence or presence of BIO.A-Control, The typical spindle fibroblast-like morphology of iPMSCs; A-BIO, The morphology of iPMSCs treated with BIO; B, iPMSCs stained with Giemsa in the absence or presence of BIO; C, The number of cells cultured in the absence or presence of BIO (0.1, 0.5, 1.0, 1.5, 2.0 µM); D, The number of iPMSCs colonies; E, Expression of PDX1, PCNA, Ngn3, TERT and C-Myc were analysed at mRNA level in the absence and presence of 1.0 and 1.5 µM BIO; F, The growth curves of iPMSCs cultured in the absence and presence of BIO (1 µM). Bar = 20 µm. * P<0.05, **, P<0.01.
Mentions: iPMSCs presented as the typical triangles and long spindle cell morphology of mesenchymal-like cells in the presence and absence of BIO (Figure 1A). Different concentrations of BIO (0, 0.1, 0.5, 1.0, 1.5, 2.0 µM) were added to the culture medium to evaluate their effects based on the morphology of iPMSCs colonies stained with Giemsa. Cell colonies were significantly denser and bigger in BIO than without BIO (Figure 1B). iPMSCs cultured in the presence of BIO (0.1, 0.5, 1.0, 1.5, 2.0 µM) for 3 and 7 days in vitro proliferated faster than that in the absence of BIO (Figure 1C). The number of cells in the presence of BIO (1.0, 1.5 µM) was elevated 1.2–2 fold compared with that in the absence of BIO. But when treated with 2.0 µM BIO, the number of iPMSCs decreased. In addition, the number of colonies was significantly increased (about 2 fold, P<0.01) in the presence of BIO (Figure 1D). The results showed that BIO could promote the proliferation of iPMSCs, and we concluded that the optimal concentrations were 1.0 and 1.5 µM. Then we detected specific pancreatic stem cell markers at the mRNA level. Expression of PDX1, Ngn3, PCNA and C-Myc showed an upward trend in iPMSCs treated with 1.0 and 1.5 µM BIO (Figure 1E) as compared with the control group. And the expression of C-Myc was clearly increased in the presence of 1.0 µM BIO compared with 1.5 µM and control. Thus, we used 1.0 µM BIO in the following experiments.

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