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 derivative cell line, PICM-31A1. PICM-31A1 cells were adapted to growth on a polymerized collagen matrix using 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 1-yr 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 that the cells expressed cytokeratin-8 and cytokeratin-18. The PICM-31FF cell line provides an in vitro model of fetal pig pancreatic exocrine cells without the complicating presence of feeder cells.
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Acknowledgements
We thank Ms. Anne Powell and Dr. Bhanu Telugu for providing adult and fetal pigs for pancreas tissue dissections.
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Care and treatment of pigs in this study were approved by the Institutional Animal Care and Use Committee of the US Department of Agriculture, Beltsville Agricultural Research Center, Beltsville, MD. Mention of trade names or commercial products in this publication is solely for the purposes of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.
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Supplementary Figure 1
Growth assay of PICM-31FF cells. PICM-31FF cells at passage 32 were cultured on polymerized collagen type I for 6 d after a 1:5 split ratio passage. Each value is ± the standard deviation (SD) of two hemocytometer counts. (DOCX 11 kb)
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Talbot, N.C., Shannon, A.E., Phillips, C.E. et al. Feeder-cell-independent culture of the pig embryonic stem cell-derived exocrine pancreatic cell line, PICM-31. In Vitro Cell.Dev.Biol.-Animal 54, 321–330 (2018). https://doi.org/10.1007/s11626-017-0218-2
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DOI: https://doi.org/10.1007/s11626-017-0218-2