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Stem Cell Reviews and Reports

, Volume 10, Issue 1, pp 31–43 | Cite as

Reprogramming of Pig Dermal Fibroblast into Insulin Secreting Cells by a Brief Exposure to 5-aza-cytidine

  • G. Pennarossa
  • S. Maffei
  • M. Campagnol
  • M. M. Rahman
  • T. A. L. Brevini
  • F. GandolfiEmail author
Article

Abstract

Large animal models provide useful data for pre-clinical research including regenerative medicine. However whereas the derivation of tissue specific stem cells has been successful. pluripotent stem cells so far have been difficult to obtain in these species. A possible alternative could be direct reprogramming but this has only been described in mouse and human. We have recently described an alternative method for reprogramming human somatic cells based on a brief demethylation step immediately followed by an induction protocol. Aim of the present paper was to determine whether this method is applicable to pig in the attempt to achieve cell reprogramming in a large animal model for the first time. Pig dermal fibroblasts were exposed to DNA methyltransferase inhibitor 5-aza-cytidine (5-aza-CR) for 18 h. After a brief recovery period, fibroblast were subjected to a three-step protocol for the induction of endocrine pancreatic differentiation that was completed after 42 days. During the process pig fibroblast rapidly lost their typical elongated form and gradually became organized in a reticular pattern that evolved into distinct cell aggregates. After a brief expression of some pluripotency genes, cells expression pattern mimicked the transition from primitive endoderm to endocrine pancreas. Not only converted cells expressed insulin but were able to release it in response to a physiological glucose challenge in vitro. Finally they were able to protect recipient mice against streptozotocin-induced diabetes. This work shows, that the conversion of a somatic cell into another, even if belonging to a different germ layer, is possible also in pig.

Keywords

Pre-clinical model Epigenetic conversion Diabetes 

Notes

Acknowledgments

Funded by NetLiPS Project ID 30190629 and Carraresi Foundation. GP was supported by Istituto Nazionale Genetica Molecolare (INGM). We thank Valentina Castiglioni for help with flow cytometry. The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception environment.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Supplementary material

12015_2013_9477_Fig7_ESM.jpg (21 kb)
Figure S1

Global methylation pattern of fibroblasts exposed to 5-aza-CR during their pancreatic differentiations. Histogram represents dot-blot signal intensity quantified by densitometric analysis using Image J analysis software (National Institutes of Health). Bars represents the mean ± SD of three independent replicates. (JPEG 20 kb)

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High Resolution Image (TIFF 225 kb)
12015_2013_9477_Fig8_ESM.jpg (15 kb)
Figure S2

Expression pattern of vimentin and pluripotency related genes in porcine fibroblasts after 5-aza-CR exposure. Untreated fibroblasts (T0) expressed high levels of vimentin, a fibroblast-specific marker. Exposure to 5-aza-CR resulted in a sharp down-regulation of vimentin accompanied by the onset of pluripotency marker expression. After 7 d of culture in endocrine pancreatic induction medium, expression of vimentin and pluripotency genes was no longer detectable. (JPEG 15 kb)

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High Resolution Image (TIFF 155 kb)
12015_2013_9477_Fig9_ESM.jpg (30 kb)
Figure S3

The effect of 5-aza-CR is reversible, and cells maintain a normal karyotype. (A)When post-5-aza-CR porcine fibroblasts were returned to standard culture medium they reverted to the initial morphology and resumed vimentin expression (Scale bar, 200 μm). (B) Within 4 d, fibroblasts completely down-regulated pluripotency-related genes and expressed vimentin at the same level measured before exposure to 5-aza-CR. (C) Cells maintained a normal karyotype for 102 d, the entire length of the experiment. (JPEG 30 kb)

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High Resolution Image (TIFF 2029 kb)
12015_2013_9477_Fig10_ESM.jpg (36 kb)
Figure S4

Expression patterns of hormone and glucose sensor genes characteristic of mature endocrine pancreatic cells in porcine epiCCs. All genes showed an expression level of physiological relevance. Ghrelin mRNA was the only one that could not be detected. (JPEG 35 kb)

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High Resolution Image (TIFF 389 kb)
12015_2013_9477_Fig11_ESM.jpg (20 kb)
Figure S5

Insulin, C-peptide, somatostatin and glucagone content in skin fibroblasts subjected to endocrine pancreatic induction. Insulin (A), C-peptide (B), somatostatin (C) and glucagon (D) were detected from day 14 of induction and increased steadily, reaching a staining intensity comparable to that obtained from fresh pig pancreatic islets with the exception of somatostatin that never exceeded the 50% of the fresh islets control. (JPEG 19 kb)

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High Resolution Image (TIFF 245 kb)
12015_2013_9477_MOESM6_ESM.doc (61 kb)
Table S1 (DOC 61 kb)
12015_2013_9477_MOESM7_ESM.doc (68 kb)
Table S2 (DOC 68 kb)

References

  1. 1.
    Ireland, J. J., Roberts, R. M., Palmer, G. H., Bauman, D. E., & Bazer, F. W. (2008). A commentary on domestic animals as dual-purpose models that benefit agricultural and biomedical research. Journal of Animal Science, 86, 2797–2805.PubMedCrossRefGoogle Scholar
  2. 2.
    Spencer, N. D., Gimble, J. M., & Lopez, M. J. (2011). Mesenchymal stromal cells: past, present, and future. Veterinary Surgery, 40, 129–139.PubMedCrossRefGoogle Scholar
  3. 3.
    Gandolfi, F., Vanelli, A., Pennarossa, G., Rahaman, M., Acocella, F., & Brevini, T. A. (2011). Large animal models for cardiac stem cell therapies. Theriogenology, 75, 1416–1425.PubMedCrossRefGoogle Scholar
  4. 4.
    Evans, M. J., Notarianni, E., Laurie, S., & Moor, R. M. (1990). Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocysts. Theriogenology, 33, 125–128.CrossRefGoogle Scholar
  5. 5.
    Piedrahita, J. A., Anderson, G. B., & BonDurant, R. H. (1990). Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines. Theriogenology, 34, 865–877.PubMedCrossRefGoogle Scholar
  6. 6.
    Brevini, T. A., Antonini, S., Pennarossa, G., & Gandolfi, F. (2008). Recent progress in embryonic stem cell research and its application in domestic species. Reproduction in Domestic Animals, 43(Suppl 2), 193–199.PubMedCrossRefGoogle Scholar
  7. 7.
    Martins-Taylor, K., & Xu, R.-H. (2010). Determinants of pluripotency: from avian, rodents, to primates. Journal of Cellular Biochemistry, 109, 16–25.PubMedGoogle Scholar
  8. 8.
    Keefer, C. L., Pant, D., Blomberg, L., & Talbot, N. C. (2007). Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Animal Reproduction Science, 98, 147–168.PubMedCrossRefGoogle Scholar
  9. 9.
    Tecirlioglu, R. T., & Trounson, A. O. (2007). Embryonic stem cells in companion animals (horses, dogs and cats): present status and future prospects. Reproduction, Fertility and Development, 19, 740–747.CrossRefGoogle Scholar
  10. 10.
    Talbot, N. C., & Blomberg, L. A. (2008). The pursuit of ES cell lines of domesticated ungulates. Stem Cell Reviews, 4, 235–254.PubMedCrossRefGoogle Scholar
  11. 11.
    Gandolfi, F., Pennarossa, G., Maffei, S., & Brevini, T. (2012). Why is it so difficult to derive pluripotent stem cells in domestic ungulates? Reproduction in Domestic Animals, 47(Suppl 5), 11–17.PubMedCrossRefGoogle Scholar
  12. 12.
    Alberio, R., Croxall, N., & Allegrucci, C. (2010). Pig epiblast stem cells depend on activin/nodal signaling for pluripotency and self-renewal. Stem Cells and Development, 19, 1627–1636.PubMedCrossRefGoogle Scholar
  13. 13.
    Esteban, M. A., Xu, J., Yang, J., et al. (2009). Generation of induced pluripotent stem cell lines from Tibetan miniature pig. Journal of Biological Chemistry, 284, 17634–17640.PubMedCrossRefGoogle Scholar
  14. 14.
    Ezashi, T., Telugu, B. P., Alexenko, A. P., Sachdev, S., Sinha, S., & Roberts, R. M. (2009). Derivation of induced pluripotent stem cells from pig somatic cells. Proceedings of the National Academy of Sciences of the United States of America, 106, 10993–10998.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Wu, Z., Chen, J., Ren, J., et al. (2009). Generation of pig induced pluripotent stem cells with a drug-inducible system. Journal of Molecular Cell Biology, 1, 46–54.PubMedCrossRefGoogle Scholar
  16. 16.
    West, F. D., Terlouw, S. L., Kwon, D. J., et al. (2010). Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells and Development, 19, 1211–1220.PubMedCrossRefGoogle Scholar
  17. 17.
    Montserrat, N., de Onate, L., Garreta, E., et al. (2012). Generation of feeder free pig induced pluripotent stem cells without Pou5f1. Cell Transplantation 21, 815-825.Google Scholar
  18. 18.
    Liu, J., Balehosur, D., Murray, B., Kelly, J. M., Sumer, H., & Verma, P. J. (2012). Generation and characterization of reprogrammed sheep induced pluripotent stem cells. Theriogenology, 77, 338–346.PubMedCrossRefGoogle Scholar
  19. 19.
    Li, Y., Cang, M., Lee, A. S., Zhang, K., & Liu, D. (2011). Reprogramming of sheep fibroblasts into pluripotency under a drug-inducible expression of mouse-derived defined factors. PLoS One, 6, e15947.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Bao, L., He, L., Chen, J., et al. (2011). Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Research, 21, 600–608.PubMedCrossRefGoogle Scholar
  21. 21.
    Sumer, H., Liu, J., Malaver-Ortega, L. F., Lim, M. L., Khodadadi, K., & Verma, P. J. (2011). NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts. Journal of Animal Science, 89, 2708–2716.PubMedCrossRefGoogle Scholar
  22. 22.
    Nagy, K., Sung, H. K., Zhang, P., et al. (2011). Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Reviews, 7, 693–702.PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    West, F. D., Uhl, E. W., Liu, Y., et al. (2011). Brief report: chimeric pigs produced from induced pluripotent stem cells demonstrate germline transmission and no evidence of tumor formation in young pigs. Stem Cells, 29, 1640–1643.PubMedCrossRefGoogle Scholar
  24. 24.
    Zhou, L., Wang, W., Liu, Y., et al. (2011). Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina. Stem Cells, 29, 972–980.PubMedCrossRefGoogle Scholar
  25. 25.
    Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–676.PubMedCrossRefGoogle Scholar
  26. 26.
    Okita, K., Ichisaka, T., & Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature, 448, 313.PubMedCrossRefGoogle Scholar
  27. 27.
    Yamanaka, S. (2009). A fresh look at iPS cells. Cell, 137, 13–17.PubMedCrossRefGoogle Scholar
  28. 28.
    Okita, K., Matsumura, Y., Sato, Y., et al. (2011). A more efficient method to generate integration-free human iPS cells. Nature Methods, 8, 409–412.Google Scholar
  29. 29.
    Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., & Hochedlinger, K. (2008). Induced pluripotent stem cells generated without viral integration. Science, 322, 945–949.PubMedCrossRefGoogle Scholar
  30. 30.
    Lengner, C. J. (2010). iPS cell technology in regenerative medicine. Annals of the New York Academy of Sciences, 1192, 38–44.PubMedCrossRefGoogle Scholar
  31. 31.
    Cohen, D. E., & Melton, D. (2011). Turning straw into gold: directing cell fate for regenerative medicine. Nature Reviews Genetics, 12, 243–252.PubMedCrossRefGoogle Scholar
  32. 32.
    Ieda, M., Fu, J.-D., Delgado-Olguin, P., et al. (2010). Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 142, 375–386.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Vierbuchen, T. (2010). Direct conversion of fibroblasts to functional neurons by defined factors. Nature, 463, 1035–1041.PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Huang, P., He, Z., Ji, S., et al. (2011). Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature, 475, 386–389.Google Scholar
  35. 35.
    Caiazzo, M., Dell/’Anno, M. T., Dvoretskova, E., et al. (2011).Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature, 476, 224–227.Google Scholar
  36. 36.
    Qiang, L., Fujita, R., Yamashita, T., et al. (2011). Directed conversion of Alzheimer’s disease patient skin fibroblasts into functional neurons. Cell, 146, 359–371.PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Pang, Z. P., Yang, N., Vierbuchen, T., et al. (2011). Induction of human neuronal cells by defined transcription factors. Nature, 476, 220–223.PubMedCentralPubMedGoogle Scholar
  38. 38.
    Nam, Y.-J., Song, K., Luo, X., et al. (2013). Reprogramming of human fibroblasts toward a cardiac fate. Proceedings of the National Academy of Sciences, 110, 5588–5593.CrossRefGoogle Scholar
  39. 39.
    Pennarossa, G., Maffei, S., Campagnol, M., Tarantini, L., Gandolfi, F., & Brevini, T. A. L. (2013). Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proceedings of the National Academy of Sciences, 110, 8948–8953.CrossRefGoogle Scholar
  40. 40.
    Bondioli, K., Ramsoondar, J., Williams, B., Costa, C., & Fodor, W. (2001). Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar. Molecular Reproduction and Development, 60, 189–195.PubMedCrossRefGoogle Scholar
  41. 41.
    Brevini, T. A., Pennarossa, G., Attanasio, L., Vanelli, A., Gasparrini, B., & Gandolfi, F. (2010). Culture conditions and signalling networks promoting the establishment of cell lines from parthenogenetic and biparental pig embryos. Stem Cell Reviews, 6, 484–495.PubMedCrossRefGoogle Scholar
  42. 42.
    Read, S. M., & Northcote, D. H. (1981). Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Analytical Biochemistry, 116, 53–64.PubMedCrossRefGoogle Scholar
  43. 43.
    Josefsen, K., Buschard, K., Sorensen, L. R., Wollike, M., Ekman, R., & Birkenbach, M. (1998). Glucose stimulation of pancreatic beta-cell lines induces expression and secretion of dynorphin. Endocrinology, 139, 4329–4336.PubMedGoogle Scholar
  44. 44.
    Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R., & McKay, R. (2001). Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science, 292, 1389–1394.PubMedCrossRefGoogle Scholar
  45. 45.
    Shi, Y., Hou, L., Tang, F., et al. (2005). Inducing embryonic stem cells to differentiate into pancreatic beta cells by a novel three-step approach with activin A and all-trans retinoic acid. Stem Cells, 23, 656–662.PubMedCrossRefGoogle Scholar
  46. 46.
    Beaujean, N., Taylor, J., Gardner, J., Wilmut, I., Meehan, R., & Young, L. (2004). Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer. Biology of Reproduction, 71, 185–193.PubMedCrossRefGoogle Scholar
  47. 47.
    Enright, B. P., Kubota, C., Yang, X., & Tian, X. C. (2003). Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2′-deoxycytidine. Biology of Reproduction, 69, 896–901.PubMedCrossRefGoogle Scholar
  48. 48.
    Jeon, B. G., Coppola, G., Perrault, S. D., Rho, G. J., Betts, D. H., & King, W. A. (2008). S-adenosylhomocysteine treatment of adult female fibroblasts alters X-chromosome inactivation and improves in vitro embryo development after somatic cell nuclear transfer. Reproduction, 135, 815–828.PubMedCrossRefGoogle Scholar
  49. 49.
    Constantinides, P. G., Jones, P. A., & Gevers, W. (1977). Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment. Nature, 267, 364–366.PubMedCrossRefGoogle Scholar
  50. 50.
    Jones, P. A. (1985). Altering gene expression with 5-azacytidine. Cell, 40, 485–486.PubMedCrossRefGoogle Scholar
  51. 51.
    Glover, T. W., Coyle-Morris, J., Pearce-Birge, L., Berger, C., & Gemmill, R. M. (1986). DNA demethylation induced by 5-azacytidine does not affect fragile X expression. American Journal of Human Genetics, 38, 309–318.PubMedCentralPubMedGoogle Scholar
  52. 52.
    Taylor, S. M., & Jones, P. A. (1979). Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell, 17, 771–779.PubMedCrossRefGoogle Scholar
  53. 53.
    Do, J. T., & Scholer, H. R. (2004). Nuclei of embryonic stem cells reprogram somatic cells. Stem Cells, 22, 941–949.PubMedCrossRefGoogle Scholar
  54. 54.
    Niwa, H. (2007). How is pluripotency determined and maintained? Development, 134, 635–646.PubMedCrossRefGoogle Scholar
  55. 55.
    Anastasia, L., Sampaolesi, M., Papini, N., et al. (2006). Reversine-treated fibroblasts acquire myogenic competence in vitro and in regenerating skeletal muscle. Cell Death and Differentiation, 13, 2042–2051.PubMedCrossRefGoogle Scholar
  56. 56.
    Davidson, S., Crowther, P., Radley, J., & Woodcock, D. (1992). Cytotoxicity of 5-aza-2′-deoxycytidine in a mammalian cell system. European Journal of Cancer, 28, 362–368.PubMedCrossRefGoogle Scholar
  57. 57.
    Wiese, C., Rolletschek, A., Kania, G., et al. (2004). Nestin expression–a property of multi-lineage progenitor cells? Cellular and Molecular Life Sciences, 61, 2510–2522.PubMedCrossRefGoogle Scholar
  58. 58.
    Ahlgren, U., Pfaff, S. L., Jessell, T. M., Edlund, T., & Edlund, H. (1997). Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells. Nature, 385, 257–260.PubMedCrossRefGoogle Scholar
  59. 59.
    Zhang, H., Wang, W. P., Guo, T., et al. (2009). The LIM-homeodomain protein ISL1 activates insulin gene promoter directly through synergy with BETA2. Journal of Molecular Biology, 392, 566–577.PubMedCrossRefGoogle Scholar
  60. 60.
    Kahan, B. W., Jacobson, L. M., Hullett, D. A., et al. (2003). Pancreatic precursors and differentiated islet cell types from murine embryonic stem cells: an in vitro model to study islet differentiation. Diabetes, 52, 2016–2024.PubMedCrossRefGoogle Scholar
  61. 61.
    Wierup, N., Svensson, H., Mulder, H., & Sundler, F. (2002). The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas. Regulatory Peptides, 107, 63–69.PubMedCrossRefGoogle Scholar
  62. 62.
    D’Amour, K. A., Bang, A. G., Eliazer, S., et al. (2006). Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nature Biotechnology, 24, 1392–1401.PubMedCrossRefGoogle Scholar
  63. 63.
    Vignjevic, S., Todorovic, V., Damjanovic, S., et al. (2012). Similar developmental patterns of ghrelin- and glucagon-expressing cells in the human pancreas. Cells, Tissues, Organs, 196, 362–373.Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • G. Pennarossa
    • 1
  • S. Maffei
    • 1
  • M. Campagnol
    • 1
  • M. M. Rahman
    • 1
  • T. A. L. Brevini
    • 1
  • F. Gandolfi
    • 1
    Email author
  1. 1.Laboratory of Biomedical Embryology - Department of Health, Animal Science and Food Safety and Center for Stem Cell ResearchUniversità degli Studi di MilanoMilanItaly

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