Exendin-4 enhances the differentiation of Wharton's jelly mesenchymal stem cells into insulin-producing cells through activation of various β-cell markers

Abstract

Background: Diabetes mellitus is a devastating metabolic disease. Generation of insulin-producing cells (IPCs) from stem cells, especially from Wharton's jelly mesenchymal stem cells (WJ-MSCs), has sparked much interest recently. Exendin-4 has several beneficial effects on MSCs and β cells. However, its effects on generation of IPCs from WJ-MSCs specifically have not been studied adequately. The purpose of this study was therefore to investigate how exendin-4 could affect the differentiation outcome of WJ-MSCs into IPCs, and to investigate the role played by exendin-4 in this differentiation process.

Methods: WJ-MSCs were isolated, characterized and then induced to differentiate into IPCs using two differentiation protocols: protocol A, without exendin-4; and protocol B, with exendin-4. Differentiated IPCs were assessed by the expression of various β-cell-related markers using quantitative RT-PCR, and functionally by measuring glucose-stimulated insulin secretion.

Results: The differentiation protocol B incorporating exendin-4 significantly boosted the expression levels of β-cell-related genes Pdx-1, Nkx2.2, Isl-1 and MafA. Moreover, IPCs generated by protocol B showed much better response to variable glucose concentrations as compared with those derived from protocol A, which totally lacked such response. Furthermore, exendin-4 alone induced early differentiation markers such as Pdx-1 and Nkx2.2 but not Isl-1, besides inducing late markers such as MafA. In addition, exendin-4 showed a synergistic effect with nicotinamide and β-mercaptoethanol in the induction of these markers.

Conclusions: Exendin-4 profoundly improves the differentiation outcome of WJ-MSCs into IPCs, possibly through the ability to induce the expression of β-cell markers.

Keywords: Diabetes mellitus; Exendin-4; Insulin-producing cells; Mesenchymal stem cells; Wharton’s jelly.

Fig. 1

a Phase-contrast image of isolated WJ-MSCs (passage 3) showing homogeneous fibroblast-like cells. Magnification: 10×; scale bar = 100 μm. b Immunophenotyping for isolated WJ-MSCs; cells were labeled with FITC-conjugated or PE-conjugated antibodies and examined by flow cytometry. The immunophenotyping profile of WJ-MSCs showed negative for CD34 and CD14 but positive for CD73, CD90 and CD105. c–h Differentiation of isolated cells down several mesenchymal lineages: c uninduced WJ-MSCs as control for adipogenic differentiation; d induced WJ-MSCs showing red staining of oil droplets using Oil Red, characteristic for successful adipogenic differentiation; e uninduced control WJ-MSCs for osteogenic differentiation experiment; f induced WJ-MSCs showing positive Alizarin Red-S staining for calcium-rich extracellular matrix, indicating successful osteogenic differentiation characteristic for MSCs; g uninduced control WJ-MSCs for the chondrogenic differentiation experiment; h induced WJ-MSCs showing blue staining by Alcian 8GX blue for sulfated proteoglycan, indicating the successful chondrogenic differentiation of isolated WJ-MSCs. Scale bar = 100 μm

Fig. 2

a–d Phase-contrast images of differentiated IPCs obtained from WJ-MSCs after induction by differentiation protocols: upon differentiation, cells lose their fibroblastic morphology and tend to aggregate forming clusters, which tend to detach and grow in suspension media, in contrast to their control WJ-MSCs which retain fibroblast-like morphology. Magnification, 20×; scale bar = 20 μm. e–h Phase-contrast images of generated IPCs stained by Dithizone (DTZ): e uninduced WJ-MSCs as a control showing negative DTZ staining; f IPCs generated by protocol A showing positive crimson red staining by DTZ; g uninduced WJ-MSCs as a control showing negative DTZ staining; h IPCs generated by protocol B showing positive crimson red staining by DTZ. Magnification 40×; scale bar = 20 μm. i In-vitro GSIS assay for differentiated IPCs derived from protocol A (without exendin-4) and protocol B (with exendin-4); insulin release in response to either 5.5 mM or 16.7 mM glucose concentrations was measured after 1 hour of incubation. It is noteworthy that control uninduced undifferentiated WJ-MSCs did not show any detectable levels of secreted insulin in response to either 5.5 mM or 16.7 mM glucose concentrations. Results presented as mean ± standard error of mean, obtained from three independent experiments. *Means are significantly different from secreted insulin levels in response to 5.5 mM glucose at p < 0.05. GSIS glucose-stimulated insulin secretion, HG high glucose, LG low glucose

Fig. 3

Gene expression analyses by qRT-PCR for several β-cell markers. a Pdx-1, b Nkx2.2, c Isl-1 and d MafA in differentiated IPCs derived from differentiation protocols A and B as compared with their control uninduced WJ-MSCs. Results are presented as mean ± standard error of mean, obtained from three independent experiments. *Means are significantly different from control uninduced WJ-MSCs at p < 0.05

Fig. 4

Gene expression analyses by qRT-PCR for cells induced by individual extrinsic factors, namely exendin-4 alone (Ex), exendin-4 plus nicotinamide (Ex + NA), and exendin-4 plus nicotinamide and β-mercaptoethanol (Ex + NA + BME), in 5 % FBS HG-DMEM. Gene expression analyses were done for several β-cell-related genes, namely a Pdx-1, b Nkx2.2, c Isl-1 and d MafA presented as relative mRNA expression levels in induced cells relative to their uninduced control counterparts. Results presented as mean ± standard error of mean for three independent experiments. *Means are significantly different from control at p < 0.05. ^#Means are significantly different from exendin-4 alone (Ex) at p < 0.05