Submitted to: In Vitro Cellular and Developmental Biology - Animal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/10/2019
Publication Date: 3/3/2019
Citation: Talbot, N.C., Shannon, A.E., Garrett, W.M. 2019. Pancreatic duct-like cell line derived from pig embryonic stem cells: expression of uroplakin genes in pig pancreatic tissue. In Vitro Cellular and Developmental Biology - Animals. 55(4):285-301.
Interpretive Summary: The PICM-31D cell line was derived from a previously isolated cell line, PICM-31, that was made from the embryonic stem cell of the pig. The original PICM-31 cells produced the digestive enzymes of the pancreas. The PICM-31D cells, in contrast, appeared to be cells that were very different in function. The report describes the characterization of the PICM-31D cells and compares its cellular feature to that of the original (parental) PICM-31 cells. The first major difference was that 31D cells grew as a continuous sheet of millions of cells, rather the as discrete colonies of 50-100 cells. Electron microscopic examination revealed that the 31D cells did not containing vesicles typical of those that are filled with digestive enzymes, instead they contained vesicles filled with mucus, typical of some of the cells that comprise the ducts of the pancreas. It was shown that the 31D cells produced various muco-proteins (mucins) by mass spectroscopy and gene expression analysis. The 31D cells also expressed 'marker' genes that were typical of cells of the ducts of the pancreas. The 31D cells therefore appear to be a cell line of mucus-secreting pancreatic ductal cells. The PICM-31D cells could be used as an in vitro (“in glass”) model of the pancreatic pathologies associated with cystic fibrosis disease in people.
Technical Abstract: The isolation of a cell line, PICM-31D, with phenotypic characteristics like pancreatic duct cells is described. The PICM-31D cell line was derived from the previously described pig embryonic stem cell-derived exocrine pancreatic cell line, PICM-31. The PICM-31D cell line was morphologically distinct from the parental cells in growing as a monolayer rather than self-assembling into multicellular acinar-like structures. The PICM-31D cells were propagated for over a year at split ratios of 1:3 to 1:10 at each passage without change in phenotype or growth rate. Electron microscopy showed the cells to be a polarized epithelium of cuboidal cells joined by tight junction-like adhesions at their apical/lateral aspect. The cells contained numerous mucus-like secretory vesicles under their apical cell membrane. Proteomic analysis of the PICM-31D's cellular proteins detected MUC1 and MUC4, consistent with mucus vesicle morphology. Gene expression analysis showed the cells expressed pancreatic ductal cell-related transcription factors such as GATA4, GATA6, HES1, HNF1A, HNF1B, ONECUT1 (HNF6), PDX1, and SOX9, but little or no pancreas progenitor cell markers such as PTF1A, NKX6-1, SOX2 or NGN3. Pancreas ductal cell-associated genes including CA2, CFTR, MUC1, MUC5B, MUC13, SHH, TFF1, KRT8 and KRT19 were expressed by the PICM-31D cells, but the exocrine pancreas marker genes, CPA1 and PLA2G1B, were not expressed by the cells. However, exocrine marker, AMY2A, was still expressed by the cells. Surprisingly, uroplakin proteins were prominent in the PICM-31D cell proteome, particularly UPK1A. Annexin A1 and A2 proteins were also relatively abundant in the cells. The expression of the uroplakin and annexin genes was detected in the cells, although only UPK1B, UPK3B, ANXA2 and ANXA4 were detected in fetal pig pancreatic duct tissue. In conclusion, the PICM-31D cell line models the mucus secreting ductal cells of the fetal pig pancreas.