The generation of islet-like insulin-producing cells from Wharton's jelly-derived mesenchymal stem cells on the PES/fish gelatin scaffold

Abstract

Diabetes Mellitus (DM) disrupts the body's capability to control blood glucose statuses. Type 1 diabetes mellitus (T1DM) arises from inadequate insulin production and is treated with insulin replacement therapy. Stem cell therapy is a hopeful treatment for T1DM that involves using adult stem cells to generate insulin-producing cells (IPCs). Mesenchymal stem cells (MSCs) are particularly advantageous for generating IPCs. The islet cells require interactions with the extracellular matrix for survival, which is lacking in conventional 2D culture systems. Natural or synthetic polymers create a supportive 3D microenvironment in tissue engineering. We aim to construct superior differentiation conditions employing polyethersulfone (PES)/Fish gelatin scaffolds to differentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) to IPCs. In this study, the PES/fish gelatin scaffold (3D) was manufactured by electrospinning, and then its biocompatibility and non-toxicity were investigated by MTT assay. After that, scaffold-supportive effects on WJ-MSCs differentiation to IPCs were studied at the gene and protein levels. After exposure to the differentiation media, 2D and 3D (PES/Fish gelatin) cultured cells were slowly aggregated and developed spherical-shaped clusters. The viability of cells was found to be comparable in both 2D and 3D cultures. The gene expression analysis showed that efficiency of differentiation was more elevated in 3D culture. Additionally, ELISA results indicated that C-peptide and insulin release were more significant in 3D than in 2D culture. In conclusion, the PES/fish gelatin scaffold is highly promising for pancreatic tissue engineering because it supports the viability, growth, and differentiation of WJ-MSCs into IPCs.

Keywords: 3D culture; Fish gelatin; Insulin-producing cells; Polyethersulfone; Scaffold; Wharton's jelly-derived mesenchymal stem cells.

Fig. 1

Isolated WJ-MSC had a positive expression for mesenchymal markers CD73, CD90, CD105, and CD166 and a negative expression for hematopoietic markers CD45, CD31 and CD34.

Fig. 2

Morphological investigation. (A) The undifferentiated WJ-MSCs exhibited a fibroblast-like spindle shape. (B) The differentiated cells into IPCs in the 2D experiment group. (C) Unseeded PES/Fish gelatin scaffold. (D) Round and spherical clusters derived from differentiated WJ-MSCs into IPCs.

Fig. 3

To assess the viability of WJ-MSCs, an MTT test was conducted. The cells were seeded on PES/Fish gelatin and cultured in 2D medium for a period of 1, 3, 5, and 7 days. The experiment was repeated three times to ensure accurate results.

Fig. 4

The real-time PCR analysis revealed that the expression of Pdx-1, insulin, glucagon, and Glut-2 genes was significantly elevated in cells cultured under 3D conditions compared to the 2D group and control.∗p < 0.05, ∗∗∗p < 0.001.

Fig. 5

Immounocytochemistry assessment of C-peptide and glucagon protein in WJ-MSCs cultivated on (A) 2D medium and (B) 3D scaffold.

Fig. 6

In vitro responses of differentiated WJ-MSCs and secretion of insulin (A) and C-peptide (B) to the various glucose concentrations. Cells were examined in the experimental (cultured on PES/Fish gelatin scaffold and 2D medium) and control groups. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.