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Establishment of a porcine pancreatic stem cell line using T-REx(™) system-inducible Wnt3a expression
Wei Han1, Xin He, Mingzhi Zhang
1College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, China; Shaanxi Stem Cell Engineering and Technology Research Center, Northwest A& F University, Yangling, 712100, China.
Objectives:
Porcine pancreatic stem cells (PSCs) are highly valuable in transplantation applications for type II diabetes. However, there are still many problems to be solved before they can be used in the clinic, such as insufficient cell number availability and low secretion level of insulin. It has been reported that Wnt3a plays pivotal roles during cell proliferation and differentiation. Here, we have aimed to establish an ideal research platform using the T-REx(™) system, to study mechanisms of Wnt3a during PSC proliferation and differentiation.
Materials And Methods:
Construction of the recombinant plasmid and cell transfection were used for establishment of a porcine PSC line. Related gene expressions were examined using quantitative real-time PCR (QRT-PCR), western blotting, immunostaining and flow cytometry. BrdU incorporation assay and cell cycle analysis were used to investigate Wnt3a roles in PSCs.
Results:
Wnt3a-expressing clones regulated by T-REx(™) were successfully obtained. Wnt3a and GFP expression were strictly regulated by Dox in a time- and dose-dependent manner. Furthermore, we found that Wnt3a-expressing porcine PSCs induced by Dox exhibited raised proliferative potential. After Dox stimulation, expression of PCNA, C-MYC and active β-catenin were higher, but were down-regulated after Dkk1 addition.
Conclusion:
We established a porcine PSC line that dynamically expressed Wnt3a, and we found that Wnt3a promoted PSC proliferative potential. This inducible expression system thus provides an important tool for further study on porcine PSC development and differentiation.
