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

  • Neil C. Talbot
  • Amy E. Shannon
  • Caitlin E. Phillips
  • Wesley M. Garrett
Article
  • 47 Downloads

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.

Keywords

Acinar Cell line Exocrine Pancreas Porcine 

Notes

Acknowledgements

We thank Ms. Anne Powell and Dr. Bhanu Telugu for providing adult and fetal pigs for pancreas tissue dissections.

Compliance with ethical standards

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.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11626_2017_218_MOESM1_ESM.docx (12 kb)
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)
11626_2017_218_MOESM2_ESM.docx (16 kb)
Supplementary Table 1 (DOCX 16 kb)
11626_2017_218_MOESM3_ESM.xlsx (186 kb)
Supplementary Data Sheet 1 (XLSX 185 kb)

References

  1. Ahn JY, Kim IY, Oh SJ, Hwang HS, Yi SS, Kim YN, Shin JH, Yoon YS, Seong JK (2014) Proteomic analysis of domestic pig pancreas during development using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Lab Anim Res 30(2):45–53.  https://doi.org/10.5625/lar.2014.30.2.45 PubMedPubMedCentralCrossRefGoogle Scholar
  2. Arias AE, Bendayan M (1993) Differentiation of pancreatic acinar cells into duct-like cells in vitro. Lab Investig 69(5):518–530PubMedGoogle Scholar
  3. Bonfanti P, Nobecourt E, Oshima M, Albagli-Curiel O, Laurysens V, Stangé G, Sojoodi M, Heremans Y, Heimberg H, Scharfmann R (2015) Ex vivo expansion and differentiation of human and mouse fetal pancreatic progenitors are modulated by epidermal growth factor. Stem Cells Dev 24(15):1766–1778.  https://doi.org/10.1089/scd.2014.0550 PubMedCrossRefGoogle Scholar
  4. Braun JP, Benard P, Burgat V, Rico AG (1983) Gamma glutamyl transferase in domestic animals. Vet Res Commun 6(1):77–90.  https://doi.org/10.1007/BF02214900 PubMedCrossRefGoogle Scholar
  5. Butler AE, Matveyenko AV, Kirakossian D, Park J, Gurlo T, Butler PC (2016) Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas. J Histotechnol 39(2):59–65.  https://doi.org/10.1080/01478885.2015.1106073 PubMedPubMedCentralCrossRefGoogle Scholar
  6. Cano DA, Soria B, Martín F, Rojas A (2014) Transcriptional control of mammalian pancreas organogenesis. Cell Mol Life Sci 71(13):2383–2402.  https://doi.org/10.1007/s00018-013-1510-2 PubMedCrossRefGoogle Scholar
  7. Caperna TJ, Blomberg le A, Garrett WM, Talbot NC (2011) Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media. In Vitro Cell Dev Biol Anim 47(3):218–233.  https://doi.org/10.1007/s11626-010-9382-3 PubMedCrossRefGoogle Scholar
  8. Deer EL, González-Hernández J, Coursen JD, Shea JE, Ngatia J, Scaife CL, Firpo MA, Mulvihill SJ (2010) Phenotype and genotype of pancreatic cancer cell lines. Pancreas 39(4):425–435.  https://doi.org/10.1097/MPA.0b013e3181c15963 PubMedPubMedCentralCrossRefGoogle Scholar
  9. Ehrhart M, Grube D, Bader MF, Aunis D, Gratzl M (1986) Chromogranin A in the pancreatic islet: cellular and subcellular distribution. J Histochem Cytochem 34(12):1673–1682.  https://doi.org/10.1177/34.12.2878021 PubMedCrossRefGoogle Scholar
  10. Gazdar AF, Chick WL, Oie HK, Sims HL, King DL, Weir GC, Lauris V (1980) Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proc Natl Acad Sci U S A 77(6):3519–3523.  https://doi.org/10.1073/pnas.77.6.3519 PubMedPubMedCentralCrossRefGoogle Scholar
  11. Githens S, Schexnayder JA, Moses RL, Denning GM, Smith JJ, Frazier ML (1994) Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures. In: In Vitro Cell Dev Biol Anim, vol 30A, pp 622–635Google Scholar
  12. Gittes GK (2009) Developmental biology of the pancreas: a comprehensive review. Dev Biol 326(1):4–35.  https://doi.org/10.1016/j.ydbio.2008.10.024 PubMedCrossRefGoogle Scholar
  13. Gomez DL, O'Driscoll M, Sheets TP, Hruban RH, Oberholzer J, McGarrigle JJ, Shamblott MJ (2015) Neurogenin 3 expressing cells in the human exocrine pancreas have the capacity for endocrine cell fate. PLoS One 10(8):e0133862.  https://doi.org/10.1371/journal.pone.0133862 PubMedPubMedCentralCrossRefGoogle Scholar
  14. Gu G, Dubauskaite J, Melton DA (2002) Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129(10):2447–2457PubMedGoogle Scholar
  15. Hall PA, Lemoine NR (1992) Rapid acinar to ductal transdifferentiation in cultured human exocrine pancreas. J Pathol 166(2):97–103.  https://doi.org/10.1002/path.1711660203 PubMedCrossRefGoogle Scholar
  16. He L, Diedrich J, Chu YY, Yates JR 3rd (2015) Extracting accurate precursor information for tandem mass spectra by RawConverter. Anal Chem 87(22):11361–11367.  https://doi.org/10.1021/acs.analchem.5b02721 PubMedPubMedCentralCrossRefGoogle Scholar
  17. Huang Y, Hui DY (1991) Cholesterol esterase biosynthesis in rat pancreatic AR42J cells. Post-transcriptional activation by gastric hormones. J Biol Chem 266(11):6720–6725PubMedGoogle Scholar
  18. Jacobsson B, Collins VP, Grimelius L, Pettersson T, Sandstedt B, Carlström A (1989) Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets. J Histochem Cytochem 37(1):31–37.  https://doi.org/10.1177/37.1.2642294 PubMedCrossRefGoogle Scholar
  19. Jessow NW, Hay RJ (1980) Characteristics of two rat pancreatic exocrine cell lines derived from transplantable tumors. In Vitro 16:212 (abstract)Google Scholar
  20. Jones EA, Clement-Jones M, James OF, Wilson DI (2001) Differences between human and mouse alpha-fetoprotein expression during early development. J Anat 198(5):555–559.  https://doi.org/10.1017/S0021878201007634 PubMedPubMedCentralCrossRefGoogle Scholar
  21. Junqueira CL, Carneiro J, Kelley RO (1992) Basic histology, 7th edn. Appleton and Lange, Norwalk, CTGoogle Scholar
  22. Kasper M, von Dorsche H, Stosiek P (1991) Changes in the distribution of intermediate filament proteins and collagen IV in fetal and adult human pancreas. I. Localization of cytokeratin polypeptides. Histochemistry 96(3):271–277.  https://doi.org/10.1007/BF00271547 PubMedCrossRefGoogle Scholar
  23. Keller A, Nesvizhskii AI, Kolker E, Aebersold R (2002) Empirical statistical model to estimate the accuracy of peptide identificationsm made by MS/MS and database search. Anal Chem 74(20):5383–5392.  https://doi.org/10.1021/ac025747h PubMedCrossRefGoogle Scholar
  24. Lee S, Hong SW, Min BH, Shim YJ, Lee KU, Lee IK, Bendayan M, Aronow BJ, Park IS (2011) Essential role of clusterin in pancreas regeneration. Dev Dyn 240(3):605–615.  https://doi.org/10.1002/dvdy.22556 PubMedCrossRefGoogle Scholar
  25. Liu N, Furukawa T, Kobari M, Tsao MS (1998) Comparative phenotypic studies of duct epithelial cell lines derived from normal human pancreas and pancreatic carcinoma. Am J Pathol 153(1):263–269.  https://doi.org/10.1016/S0002-9440(10)65567-8 PubMedPubMedCentralCrossRefGoogle Scholar
  26. Masamune A, Satoh M, Kikuta K, Suzuki N, Shimosegawa T (2003) Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization. World J Gastroenterol 9(12):2751–2758.  https://doi.org/10.3748/wjg.v9.i12.2751 PubMedPubMedCentralCrossRefGoogle Scholar
  27. Mato E, Lucas M, Petriz J, Gomis R, Novials A (2009) Identification of a pancreatic stellate cell population with properties of progenitor cells: new role for stellate cells in the pancreas. Biochem J 421(2):181–191.  https://doi.org/10.1042/BJ20081466 PubMedCrossRefGoogle Scholar
  28. Maurer M, Müller AC, Parapatics K, Pickl WF, Wagner C, Rudashevskaya EL, Breitwieser FP, Colinge J, Garg K, Griss J, Bennett KL, Wagner SN (2014) Comprehensive comparative and semiquantitative proteome of a very low number of native and matched epstein-barr-virus-transformed B lymphocytes infiltrating human melanoma. J Proteome Res 13(6):2830–2845.  https://doi.org/10.1021/pr401270y PubMedCrossRefGoogle Scholar
  29. Meek J, Adamson ED (1985) Transferrin in foetal and adult mouse tissues: synthesis, storage and secretion. J Embryol Exp Morphol 86:205–218PubMedGoogle Scholar
  30. Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB, Maechler P (2004) Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 145(2):667–678.  https://doi.org/10.1210/en.2003-1099 PubMedCrossRefGoogle Scholar
  31. Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75(17):4646–4658.  https://doi.org/10.1021/ac0341261 PubMedCrossRefGoogle Scholar
  32. Pagliuca FW, Melton DA (2013) How to make a functional β-cell. Development 140(12):2472–2483.  https://doi.org/10.1242/dev.093187 PubMedPubMedCentralCrossRefGoogle Scholar
  33. Powers AC, Efrat S, Mojsov S, Spector D, Habener JF, Hanahan D (1990) Proglucagon processing similar to normal islets in pancreatic alpha-like cell line derived from transgenic mouse tumor. Diabetes 39(4):406–414.  https://doi.org/10.2337/diab.39.4.406 PubMedCrossRefGoogle Scholar
  34. Quinn AR, Blanco CL, Perego C, Finzi G, La Rosa S, Capella C, Guardado-Mendoza R, Casiraghi F, Gastaldelli A, Johnson M, Dick EJ Jr, Folli F (2012) The ontogeny of the endocrine pancreas in the fetal/newborn baboon. J Endocrinol 214: 289–299, 3, DOI:  https://doi.org/10.1530/JOE-12-0070
  35. Ravassard P, Hazhouz Y, Pechberty S, Bricout-Neveu E, Armanet M, Czernichow P, Scharfmann R (2011) A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion. J Clin Invest 121(9):3589–3597.  https://doi.org/10.1172/JCI58447 PubMedPubMedCentralCrossRefGoogle Scholar
  36. Riopel M, Li J, Fellows GF, Goodyer CG, Wang R (2014) Ultrastructural and immunohistochemical analysis of the 8-20 week human fetal pancreas. Islets 6(4):e982949.  https://doi.org/10.4161/19382014.2014.982949 PubMedPubMedCentralCrossRefGoogle Scholar
  37. Schaffer AE, Taylor BL, Benthuysen JR, Liu J, Thorel F, Yuan W, Jiao Y, Kaestner KH, Herrera PL, Magnuson MA, May CL, Sander M (2013) Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic beta cell identity. PLoS Genet 9(1):e1003274.  https://doi.org/10.1371/journal.pgen.1003274 PubMedPubMedCentralCrossRefGoogle Scholar
  38. Shih HP, Kopp JL, Sandhu M, Dubois CL, Seymour PA, Grapin-Botton A, Sander M (2012) A Notch-dependent molecular circuitry initiates pancreatic endocrine and ductal cell differentiation. Development 139(14):2488–2499.  https://doi.org/10.1242/dev.078634 PubMedPubMedCentralCrossRefGoogle Scholar
  39. Skelin M, Rupnik M, Cencic A (2010) Pancreatic beta cell lines and their applications in diabetes mellitus research. ALTEX 27(2):105–113PubMedCrossRefGoogle Scholar
  40. Sorenson RL, Stout LE, Brelje TC, Van Pilsum JF, McGuire DM (1995) Evidence for the role of pancreatic acinar cells in the production of ornithine and guanidinoacetic acid by L-arginine:glycine amidinotransferase. Pancreas 10(4):389–394.  https://doi.org/10.1097/00006676-199505000-00011 PubMedCrossRefGoogle Scholar
  41. Su Y, Jono H, Misumi Y, Senokuchi T, Guo J, Ueda M, Shinriki S, Tasaki M, Shono M, Obayashi K, Yamagata K, Araki E, Ando Y (2012) Novel function of transthyretin in pancreatic alpha cells. FEBS Lett 586(23):4215–4222.  https://doi.org/10.1016/j.febslet.2012.10.025 PubMedCrossRefGoogle Scholar
  42. Talbot NC, Caperna TJ (1998) Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver. In: In Vitro Cell Dev Biol Anim, vol 34A, pp 785–798Google Scholar
  43. Talbot NC, Paape MJ (1996) Continuous culture of pig tissue-derived macrophages. Methods Cell Sci 18(4):315–327.  https://doi.org/10.1007/BF00127909 CrossRefGoogle Scholar
  44. Talbot NC, Pursel VG, Rexroad CE Jr, Caperna TJ, Powell AM, Stone RT (1994) Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells. In: In Vitro Cell Dev Biol Anim, vol 30A, pp 851–858Google Scholar
  45. Talbot NC, Rexroad CE Jr, Pursel VG, Powell AM, Nel ND (1993) Culturing the epiblast cells of the pig blastocyst. In: In Vitro Cell Dev Biol Anim, vol 29A, pp 543–554Google Scholar
  46. Talbot NC, Shannon AE, Phillips CE, Garrett WM (2017) Derivation and characterization of a pig embryonic-stem-cell-derived exocrine pancreatic cell line. Pancreas 46(6):789–800.  https://doi.org/10.1097/MPA.0000000000000836 PubMedCrossRefGoogle Scholar
  47. Tezel E, Nagasaka T, Tezel G, Kaneko T, Takasawa S, Okamoto H, Nakao A (2004) REG I as a marker for human pancreatic acinoductular cells. Hepato-Gastroenterology 51(55):91–96PubMedGoogle Scholar
  48. Ulrich AB, Schmied BM, Standop J, Schneider MB, Pour PM (2002) Pancreatic cell lines: a review. Pancreas 24(2):111–120.  https://doi.org/10.1097/00006676-200203000-00001 PubMedCrossRefGoogle Scholar
  49. Wang G, Rajpurohit SK, Delaspre F, Walker SL, White DT, Ceasrine A, Kuruvilla R, Li RJ, Shim JS, Liu JO, Parsons MJ, Mumm JS (2015) First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass. elife 4.  https://doi.org/10.7554/eLife.08261
  50. Westermark GT, Westermark P (2008) Transthyretin and amyloid in the islets of Langerhans in type-2 diabetes. Exp Diabetes Res 2008(1):429274–429277.  https://doi.org/10.1155/2008/429274 PubMedPubMedCentralGoogle Scholar
  51. Wiśniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362.  https://doi.org/10.1038/nmeth.1322 PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  1. 1.Animal Biosciences and Biotechnology LaboratoryAgricultural Research ServiceBeltsvilleUSA
  2. 2.ABBLUSDABeltsvilleUSA

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