title: Establishment of a porcine pancreatic stem cell line using T-REx(™) system-inducible Wnt3a expression.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: College 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.
authors.forename: Wei
authors.initials: W
authors.lastname: Han
authors.forename: Xin
authors.initials: X
authors.lastname: He
authors.forename: Mingzhi
authors.initials: M
authors.lastname: Zhang
authors.forename: Shuxian
authors.initials: S
authors.lastname: Hu
authors.forename: Fen
authors.initials: F
authors.lastname: Sun
authors.forename: Lipeng
authors.initials: L
authors.lastname: Ren
authors.forename: Jinlian
authors.initials: J
authors.lastname: Hua
authors.forename: Sha
authors.initials: S
authors.lastname: Peng
mesh_terms: D000818:Animals; D060846:Cell Engineering; D002460:Cell Line / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D004318:Doxycycline / Q000494:pharmacology; D018703:Founder Effect; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D010179:Pancreas / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013552:Swine; D014162:Transfection; D060509:Wnt3A Protein / Q000235:genetics* / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D018809:Proliferating Cell Nuclear Antigen; D016271:Proto-Oncogene Proteins c-myc; D060509:Wnt3A Protein; D051176:beta Catenin; D049452:Green Fluorescent Proteins; D004318:Doxycycline