Abstract
Type 1 Diabetes Mellitus (T1DM) is a chronic disease that leads to loss of insulin secreting β-cells, causing high levels of blood glucose. Exogenous insulin administration is not sufficient to mimic the normal function of β-cells and, consequently, diabetes mellitus often progresses and can lead to major chronic complications and morbidity. The physiological control of glucose levels can only be restored by replacing the β-cell mass.
We recently developed a new strategy that allows for epigenetic conversion of dermal fibroblasts into insulin-secreting cells (EpiCC), using a brief exposure to the demethylating agent 5-aza-cytidine (5-aza-CR), followed by a pancreatic induction protocol. This method has notable advantages compared to the alternative available procedures and may represent a promising tool for clinical translation as a therapy for T1DM. However, a thought evaluation of its therapeutic safety and efficacy is mandatory to support preclinical studies based on EpiCC treatment.
We here report the data obtained using human fibroblasts isolated from diabetic and healthy individuals, belonging the two genders. EpiCC were injected into 650 diabetic severe combined immunodeficiency (SCID) mice and demonstrated to be able to restore and maintain glycemic levels within the physiological range. Cells had the ability to self-regulate and not to cause hypoglycemia, when transplanted in healthy animals. Efficacy tests showed that EpiCC successfully re-established normoglycemia in diabetic mice, using a dose range that appeared clinically relevant to the concentration 0.6 × 106 EpiCC. Necropsy and histopathological investigations demonstrated the absence of malignant transformation and cell migration to organs and lymph nodes.
The present preclinical study demonstrates safety and efficacy of human EpiCC in diabetic mice and supports the use of epigenetic converted cells for regenerative medicine of diabetes mellitus.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Alejandro R, Barton FB, Hering BJ, Wease S (2008) 2008 update from the collaborative islet transplant registry. Transplantation 86(12):1783–1788
Brevini TA, Pennarossa G, Antonini S, Paffoni A, Tettamanti G, Montemurro T, Radaelli E, Lazzari L, Rebulla P, Scanziani E, de Eguileor M, Benvenisty N, Ragni G, Gandolfi F (2009) Cell lines derived from human parthenogenetic embryos can display aberrant centriole distribution and altered expression levels of mitotic spindle check-point transcripts. Stem Cell Rev 5(4):340–52
Brevini TA, Pennarossa G, Rahman MM, Paffoni A, Antonini S, Ragni G, deEguileor M, Tettamanti G, Gandolfi F (2014) Morphological and molecular changes of human Granulosa cells exposed to 5-Azacytidine and addressed toward muscular differentiation. Stem Cell Rev 10(5):633–642
Brevini TA, Pennarossa G, Maffei S, Zenobi A, Gandolfi F (2016) Epigenetic conversion as a safe and simple method to obtain insulin-secreting cells from adult skin fibroblasts. J Vis Exp: JoVE 109:e53880
Brevini T, Manzoni E, Gandolfi F (2018) Methylation mechanisms and biomechanical effectors controlling cell fate. Reprod Fertil Dev 30:64–72
Butler AE, Galasso R, Meier JJ, Basu R, Rizza RA, Butler PC (2007) Modestly increased beta cell apoptosis but no increased beta cell replication in recent-onset type 1 diabetic patients who died of diabetic ketoacidosis. Diabetologia 50(11):2323–2331
Chandrakanthan V, Yeola A, Kwan JC, Oliver RA, Qiao Q, Kang YC, Zarzour P, Beck D, Boelen L, Unnikrishnan A, Villanueva JE, Nunez AC, Knezevic K, Palu C, Nasrallah R, Carnell M, Macmillan A, Whan R, Yu Y, Hardy P, Grey ST, Gladbach A, Delerue F, Ittner L, Mobbs R, Walkley CR, Purton LE, Ward RL, Wong JW, Hesson LB, Walsh W, Pimanda JE (2016) PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells. Proc Natl Acad Sci U S A 113(16):E2306–E2315
Gepts W (1965) Pathologic anatomy of the pancreas in juvenile diabetes mellitus. Diabetes 14(10):619–633
Glover TW, Coyle-Morris J, Pearce-Birge L, Berger C, Gemmill RM (1986) DNA demethylation induced by 5-azacytidine does not affect fragile X expression. Am J Hum Genet 38(3):309–318
Gradwohl G, Dierich A, LeMeur M, Guillemot F (2000) neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A 97(4):1607–1611
Halban PA, German MS, Kahn SE, Weir GC (2010) Current status of islet cell replacement and regeneration therapy. J Clin Endocrinol Metab 95(3):1034–1043
Harris DM, Hazan-Haley I, Coombes K, Bueso-Ramos C, Liu J, Liu Z, Li P, Ravoori M, Abruzzo L, Han L, Singh S, Sun M, Kundra V, Kurzrock R, Estrov Z (2011) Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells. PLoS One 6(6):e21250
Jones PA (1985a) Altering gene expression with 5-azacytidine. Cell 40(3):485–486
Jones PA (1985b) Effects of 5-azacytidine and its 2′-deoxyderivative on cell differentiation and DNA methylation. Pharmacol Ther 28(1):17–27
Jones PA, Taylor SM (1981) Hemimethylated duplex DNAs prepared from 5-azacytidine-treated cells. Nucleic Acids Res 9(12):2933–2947
Jones PA, Taylor SM, Wilson VL (1983) Inhibition of DNA methylation by 5-azacytidine. Recent Results Cancer Res 84:202–211
Jonsson J, Carlsson L, Edlund T, Edlund H (1994) Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371(6498):606–609
Keymeulen B, Ling Z, Gorus FK, Delvaux G, Bouwens L, Grupping A, Hendrieckx C, Pipeleers-Marichal M, Van Schravendijk C, Salmela K, Pipeleers DG (1998) Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: graft and recipients characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft. Diabetologia 41(4):452–459
Keymeulen B, Gillard P, Mathieu C, Movahedi B, Maleux G, Delvaux G, Ysebaert D, Roep B, Vandemeulebroucke E, Marichal M, In’t Veld P, Bogdani M, Hendrieckx C, Gorus F, Ling Z, van Rood J, Pipeleers D (2006) Correlation between beta cell mass and glycemic control in type 1 diabetic recipients of islet cell graft. Proc Natl Acad Sci U S A 103(46):17444–17449
Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, Young H, Richardson M, Smart NG, Cunningham J, Agulnick AD, D’Amour KA, Carpenter MK, Baetge EE (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol 26(4):443–452
Lardon J, De Breuck S, Rooman I, Van Lommel L, Kruhoffer M, Orntoft T, Schuit F, Bouwens L (2004) Plasticity in the adult rat pancreas: transdifferentiation of exocrine to hepatocyte-like cells in primary culture. Hepatology 39(6):1499–1507
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(5520):1389–1394
Mathers CD, Loncar D (2006) Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 3(11):e442
Mathews V, Hanson PT, Ford E, Fujita J, Polonsky KS, Graubert TA (2004) Recruitment of bone marrow-derived endothelial cells to sites of pancreatic beta-cell injury. Diabetes 53(1):91–98
Matsuoka TA, Artner I, Henderson E, Means A, Sander M, Stein R (2004) The MafA transcription factor appears to be responsible for tissue-specific expression of insulin. Proc Natl Acad Sci U S A 101(9):2930–2933
Mirakhori F, Zeynali B, Kiani S, Baharvand H (2015) Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells. Cell J 17(1):153–158
Nathan DM, Genuth S, Lachin J, Cleary P, Crofford O, Davis M, Rand L, Siebert C (1993) The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 329(14):977–986
Nicholas CR, Kriegstein AR (2010) Regenerative medicine: cell reprogramming gets direct. Nature 463(7284):1031–1032
Nombela-Arrieta C, Ritz J, Silberstein LE (2011) The elusive nature and function of mesenchymal stem cells. Nat Rev Mol Cell Biol 12(2):126–131
Pennarossa G, Maffei S, Campagnol M, Tarantini L, Gandolfi F, Brevini TA (2013) Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proc Natl Acad Sci U S A 110(22):8948–8953
Pennarossa G, Maffei S, Campagnol M, Rahman MM, Brevini TA, Gandolfi F (2014) Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine. Stem Cell Rev 10(1):31–43
Pennarossa G, Santoro R, Manzoni E, Pesce M, Gandolfi F, Brevini T (2018) Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of low-stiffness substrate. Stem Cell Rev Rep. https://doi.org/10.1007/s12015-017-9799-0
Pinho AV, Rooman I, Reichert M, De Medts N, Bouwens L, Rustgi AK, Real FX (2011) Adult pancreatic acinar cells dedifferentiate to an embryonic progenitor phenotype with concomitant activation of a senescence programme that is present in chronic pancreatitis. Gut 60(7):958–966
Pipeleers D, Ling Z (1992) Pancreatic beta cells in insulin-dependent diabetes. Diabetes Metab Rev 8(3):209–227
Ryan EA, Lakey JR, Rajotte RV, Korbutt GS, Kin T, Imes S, Rabinovitch A, Elliott JF, Bigam D, Kneteman NM, Warnock GL, Larsen I, Shapiro AM (2001) Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50(4):710–719
Ryan EA, Paty BW, Senior PA, Bigam D, Alfadhli E, Kneteman NM, Lakey JR, Shapiro AM (2005) Five-year follow-up after clinical islet transplantation. Diabetes 54(7):2060–2069
Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343(4):230–238
Sui L, Mfopou JK, Chen B, Sermon K, Bouwens L (2013) Transplantation of human embryonic stem cell-derived pancreatic endoderm reveals a site-specific survival, growth, and differentiation. Cell Transplant 22(5):821–830
Taylor SM, Jones PA (1982) Changes in phenotypic expression in embryonic and adult cells treated with 5-azacytidine. J Cell Physiol 111(2):187–194
Weir GC, Bonner-Weir S (2013) Islet beta cell mass in diabetes and how it relates to function, birth, and death. Ann N Y Acad Sci 1281:92–105
Ziegler AG, Nepom GT (2010) Prediction and pathogenesis in type 1 diabetes. Immunity 32(4):468–478
Acknowledgements
This work was funded by European Foundation for the Study of Diabetes (EFSD) and by Carraresi Foundation. The Authors are members of the COST Action CA16119 In vitro 3-D total cell guidance and fitness (CellFit) and the COST Action CM1406 Epigenetic Chemical Biology (EPICHEM).
Conflict of Interest
The authors declare no conflict of interest in relation to this article.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer International Publishing AG
About this chapter
Cite this chapter
Brevini, T.A.L., Pennarossa, G., Manzoni, E.F.M., Gandolfi, F. (2018). Safety and Efficacy of Epigenetically Converted Human Fibroblasts Into Insulin-Secreting Cells: A Preclinical Study. In: Turksen, K. (eds) Cell Biology and Translational Medicine, Volume 1. Advances in Experimental Medicine and Biology(), vol 1079. Springer, Cham. https://doi.org/10.1007/5584_2018_172
Download citation
DOI: https://doi.org/10.1007/5584_2018_172
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-93866-0
Online ISBN: 978-3-319-93867-7
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)