Studies on differentiation of mouse GS-2 ES-cells to pancreatic β-islet-like cells and their functional maturation status

Abstract

Embryonic stem cells (ES-cells) are an excellent source for generating insulin-producing β-islet cells for potential use in the management of diabetes mellitus. Although many protocols have been developed to promote the differentiation of ESCs into β-islet cells, they are limited in terms of (i) low efficiency of differentiation and (ii) generation of functionally immature β-islet cells with inefficient glucose stimulated insulin secretion (GSIS). The present study is aimed at understanding the differentiation and functional maturity of pancreatic β-islet cell. Earlier in our lab EGFP-expressing transgenic mouse ‘GS-2’ ES-cell line was derived (Singh et al., 2012). Through the embryoid body (EB) formation and differentiation method (day 2 through 21), we obtained spontaneous differentiation of GS-2 ES-cells to three germ-lineage cell types, with the expression of molecular markers of definitive endoderm (DE; sox17) and hepatic lineage (afp). However, we were unable to detect the expression of markers of pancreatic progenitors (pdx1) and β-islet cells (ins2). By improvising the spontaneous differentiation protocol i.e. increasing cell seeding density for EB, we were able to detect expression of pancreatic progenitor marker, pdx-1. However, we were still unable to detect expression of β-islet marker ins1 and ins2. In view of this, we employed an induction protocol by inclusion of laminin, nicotinamide and insulin (Wobus et al., 2006). This resulted in the differentiation of GS-2 ES-cells to DE-like cells by day 14, followed by the appearance of pancreatic progenitor-like clusters by day 21 and significantly, β-islet-like clusters by day 33. This sequential appearance of progenitors and β-islet cells was accompanied by the expression of pdx1, ins1and ins2. However, expression of more important β-islet marker ins1 appeared to be low. Also, we were unable to detect expression of glucose transporter, glut-2 indicating, this model system may not be suitable for testing molecules to improve GSIS or functional maturity. Therefore, we found an alternative model system, Islet Cell Aggregates (ICAs) from day1-2 old neonatal mice that are considered functionally immature or have inefficient GSIS. Sirtuin-1 activator SRT1720 was found as the molecule to improve functional maturity of immature β-islet cells. When functionally immature β-islet cells from day1-2 old neonatal mice were treated with SRT1720 (5μM) for 24 hours, their GSIS improved by 1.5 fold. We envisage that this molecule may be used to improve functional immaturity of PSC-derived functionally-immature β-islet cells