Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

Authors: Talbot, Neil; Shannon, Amy; Phillips, Caitlin; Garrett, Wesley

Submitted to: In Vitro Cellular and Developmental Biology - Animal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/22/2017
Publication Date: 2/18/2018
Citation: Talbot, N.C., Shannon, A.E., Phillips, C., Garrett, W.M. 2018. Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31. In Vitro Cellular and Developmental Biology - Animals. 54:321-330.

Interpretive Summary: A pig embryonic stem cell-derived pancreatic cell line that does not require “feeder-cells” for their growth and maintenance of pancreas exocrine function was established and characterized in comparison to its cell line of origin, PICM-31. The new cell line, PICM-31FF, grew without the aid of feeder-cells after a period of adaptation to growing on a biological surface made of collagen. Like the parental PICM-31 cell line, the PICM-31FF cells were small and grew relatively slowly. Electron microscopic examination confirmed that the cells contained secretory granules similar to the cells of the pancreas that secrete digestive enzymes. The PICM-31FF cells were shown to express genes for digestive enzymes, and the secreted enzymes themselves were also identified by mass spectroscopy. The PICM-31FF cells also expressed so-called 'marker' genes indicative of the exocrine pancreas tissue of the body. If the PICM-31FF cells could be induced to become beta-cells, the pancreas cells in the body that make insulin, the cell line might be a source of insulin for diabetic people. The PICM-31FF cell line can be used to study and develop useful genetic metabolic changes in pigs, and their growth without the necessity for contact with feeder-cells simplifies the culture system so that it is more useful for modeling pancreas cells outside of the body.

Technical Abstract: The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. PICM-31A1 cells were adapted to growth on a polymerized collagen matrix using STO-feeder-cell conditioned medium and were designated PICM-31FF. Like the parental cells, the PICM-31FF cells were small and grew relatively slowly in closely knit colonies that eventually coalesced into a continuous monolayer. The PICM-31FF cells were extensively cultured; 40 passages at 1:2, 1:3 and finally 1:5 split ratios over a one year period. Ultrastructure analysis showed the cells' epithelial morphology and revealed that they retained their secretory granules typical of pancreas acinar cells. The cells maintained their expression of digestive enzymes, including carboxypeptidase A1 (CPA1), amylase 2A (AMY2A) and phospholipase A2 (PLA2G1B). Alpha-fetoprotein (AFP), a fetal cell marker, continued to be expressed by the cells as was the pancreas alpha-cell-associated gene, transthyretin. Several pancreas-associated developmental genes were also expressed by the cells, including pancreatic and duodenal homeobox 1 (PDX1) and pancreas specific transcription factor, 1a (PTF1A). Proteomic analysis of cellular proteins confirmed the cells' production of digestive enzymes and showed the cells expressed cytokeratin-8 and -18. The PICM-31FF cell line provides an in vitro model of fetal pig pancreatic exocrine cells without the complicating presence of STO feeder-cells.