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


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.


Pre-clinical model Epigenetic conversion Diabetes 



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

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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)

12015_2013_9477_MOESM5_ESM.tif (245 kb)
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)


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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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