The effect of epigenetic factors on differentiation of pancreatic progenitor cells into insulin producing cells

T. Koblas, I. Leontovyc, K. Zacharovova, Z. Berkova, P. Girman, J. Kriz, F. Saudek
Institute for Clinical and Experimental Medicine, Praha, Czech Republic

Objective: Differentiation of pancreatic progenitors into insulin-producing beta-cells is regulated by various transcription factors. The expression of these genes requires an accessible DNA in a form of active euchromatin structure. The structure of chromatin and DNA is regulated by various epigenetic modifications. In our study we have evaluated the effect of selected epigenetic factors on differentiation of human non-endocrine cells into insulin producing cells.

Methods: Human non-endocrine cells obtained from islet-depleted pancreatic tissue (n=5) were cultured for 4 days in DMEM medium containing 0,5% albumin, 1% ITS, bFGF, EGF,neonatal fibroblasts conditioned medium and various combinations of epigenetic factors such as DNA methyltransferase inhibitor 5-Aza-2'-Deoxycytidine (DAC), G9a histone methyl transferase inhibitor BIX01294 and histone deacetylase inhibitors sodium butyrate, Scriptaid and MC1568. Afterwards, cells werecultured for 3 days in CMRL medium containing 0,5% albumin, 0,1% ITS, SP600125,SB216763, forskolin, fibronectin, exendin-4, IGF-1, and nicotinamide.

Results: Non-endocrine cells formed islet-likecell clusters (ILCCs) containing mainly cytokeratin-19 positive cells within 2 days of cultivation. After the cultivation with epigenetic factors and further differentiation the highest number of C-peptide positive cells (10.3±2.9%) as well as glucagon positive cells (7.2±2.8%) was achieved in a sample supplemented with a combination of DAC, BIX01294 and MC1568. Control sample treated with no any of epigenetic factors had significantly lower number of C-peptide positive cells (2.5 ±1.6%). In response to glucose stimulation (5 vs.20 mM) DAC, BIX01294 and MC1568 treated ILCCs secreted increased amount of C-peptide (0.47 vs 1.05 pmol C-peptide/µgDNA).

Conclusions: A combination of epigenetic factors 5-Aza-2'-Deoxycytidine, BIX01294 and MC1568 significantly improves reproducible differentiation of non-endocrine pancreatic cells into insulin producing cells. This work was supported by a grant:NS/9712-4/2008 IGA of the Ministry of Health, CR.